Microbial Iron Metabolism: A Comprehensive Treatise

MICROBIAL IRON METABOLISM

A COMPREHENSIVE TREATISE

J.B. Neilands

DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA, BERKELEY, CALIFORNIA

Table of Contents

Cover image

Title page

CONTRIBUTORS

Copyright

LIST OF CONTRIBUTORS

PREFACE

PART I: INTRODUCTION

Chapter 1: IRON AND ITS ROLE IN MICROBIAL PHYSIOLOGY

Publisher Summary

I INTRODUCTION

II HISTORICAL BACKGROUND

III BIOGEOCHEMISTRY OF IRON

IV SOME PHYSICAL AND CHEMICAL PROPERTIES OF IRON

V IRON CONTENT OF MICROORGANISMS

VI IRON LIGAND ATOMS IN MICROORGANISMS AND THEIR FUNCTION

VII LIFE WITHOUT IRON AND FUNCTIONAL REPLACEMENTS FOR IRON

VIII ASPECTS OF THE COMPARATIVE BIOCHEMISTRY OF IRON METABOLISM

Chapter 2: METABOLISM IN IRON-LIMITED GROWTH

Publisher Summary

I INTRODUCTION

II IRON-LIMITED GROWTH

III PRACTICAL ASPECTS OF IRON LIMITATION

IV EFFECTS OF IRON DEFICIENCY AND IRON LIMITATION

V SUMMARY

ACKNOWLEDGMENTS

PART II: TRANSPORT, BIOSYNTHESIS, AND STORAGE

Chapter 3: IRON TRANSPORT IN THE ENTERIC BACTERIA

Publisher Summary

I INTRODUCTION

II IRON TRANSPORT SYSTEMS IN ESCHERICHIA COLI

III IRON TRANSPORT SYSTEMS IN

IV IRON TRANSPORT SYSTEMS IN

V DISCUSSION

Chapter 4: IRON TRANSPORT IN GRAM-POSITIVE AND ACID-FAST BACILLI

Publisher Summary

I Introduction

II PRODUCTION OF IRON-CHELATING AGENTS BY Bacillus SPECIES

III IRON TRANSPORT IN Bacillus megaterium

IV IRON TRANSPORT IN Bacillus subtilis

V IRON TRANSPORT IN Mycobacterium smegmatis

VI SUMMARY*

ACKNOWLEDGMENTS

Chapter 5: BIOSYNTHESIS AND MECHANISM OF ACTION OF HYDROXAMATE-TYPE SIDEROCHROMES

Publisher Summary

I INTRODUCTION

II HADACIDIN

III FERRICHROME

IV RHODOTORULIC ACID

V ASPERGILLIC ACID

VI MYCOBACTIN

VII REGULATION OF HYDROXAMATE SYNTHESIS

VIII HYDROXAMIC ACIDS AND IRON TRANSPORT

ACKNOWLEDGMENT

Chapter 6: BIOSYNTHESIS OF HEME

Publisher Summary

I INTRODUCTION

II HEME CONTENT OF VARIOUS MICROORGANISMS

III PATHWAY OF HEME SYNTHESIS

IV REGULATION OF MICROBIAL HEME SYNTHESIS

ACKNOWLEDGMENTS

Chapter 7: FERRITIN AND IRON METABOLISM IN PHYCOMYCES

Publisher Summary

I INTRODUCTION

II PURIFICATION AND PROPERTIES OF PHYCOMYCES FERRITIN

III INDUCTION OF FERRITIN SYNTHESIS BY IRON

IV FERRITIN SYNTHESIS AND LOCALIZATION IN SPORES

V FERRITIN AND IRON METABOLISM IN GERMINATING SPORES

ACKNOWLEDGMENTS

PART III: IRON ENZYMES AND PROTEINS

Chapter 8: FERREDOXIN AND RUBREDOXIN

Publisher Summary

I INTRODUCTION

II HISTORICAL BACKGROUND

III BIOLOGICAL ROLES

IV CHEMICAL PROPERTIES OF IRON-SULFUR PROTEINS

V SOME GENERAL THOUGHTS ABOUT IRON-SULFUR ELECTRON CARRIERS

ACKNOWLEDGMENT

Chapter 9: SURVEY OF NITROGENASE AND ITS EPR PROPERTIES

Publisher Summary

I INTRODUCTION

II ISOLATION AND PURIFICATION OF NITROGENASE COMPONENTS

III PHYSICOCHEMICAL PROPERTIES OF NITROGENASE COMPONENTS

IV CATALYTIC ACTIVITY OF NITROGENASE

V EPR STUDIES OF NITROGENASE AND ITS COMPONENTS

VI MECHANISM OF N2 REDUCTION

ACKNOWLEDGMENTS

Chapter 10: THE NITROGEN FIXATION (NIF) OPERON(S) OF KLEBSIELLA PNEUMONIAE

Publisher Summary

I INTRODUCTION

II THE CLUSTER OF NIF GENES NEAR HIS

III BIOCHEMICAL EVIDENCE FOR NITROGENASE GENES NEAR HIS

IV NIF– MUTATIONS UNLINKED TO HIS

V GENETIC REGULATION OF NIF

VI TRANSFER OF NIF TO E. COLI AND POTENTIAL FOR GENETIC ENGINEERING

Chapter 11: HYDROGENASE

Publisher Summary

I BACKGROUND

II DISTRIBUTION OF HYDROGENASE

III ROLE OF HYDROGENASE IN MICROBIAL METABOLISM

IV NUTRITIONAL STUDIES ON HYDROGEN METABOLISM

V ASSAYS OF HYDROGENASE

VI PURIFICATION AND PROPERTIES

VII MECHANISM OF HYDROGENASE CATALYSIS

ACKNOWLEDGMENT

Chapter 12: GLUTAMATE SYNTHASE

Publisher Summary

I INTRODUCTION

II DISCOVERY

III DISTRIBUTION

IV REGULATION OF GLUTAMATE SYNTHASE LEVELS

V MUTANTS LACKING GLUTAMATE SYNTHASE

VI KINETIC PARAMETERS

VII GLUTAMATE SYNTHASE FROM E. COLI

VII CONCLUSIONS

Chapter 13: NONHEME IRON IN RESPIRATORY CHAINS

Publisher Summary

I INTRODUCTION

II METHODOLOGY FOR STUDY OF RESPIRATORY CHAIN-LINKED NONHEME IRON

III NONHEME IRON IN RESPIRATORY CHAINS OF EUKARYOTIC CELLS

IV NONHEME IRON IN THE RESPIRATORY CHAINS OF PROKARYOTIC CELLS

ACKNOWLEDGMENT

Chapter 14: CYTOCHROMES

Publisher Summary

I GENERAL SURVEY

II CYTOCHROME A AND CYTOCHROME OXIDASE

III CYTOCHROME B

IV CYTOCHROME C

V HEME d-BEARING CYTOCHROME

Chapter 15: HYDROPEROXIDASES

Publisher Summary

I INTRODUCTION

II YEAST CYTOCHROME c PEROXIDASE

III PSEUDOMONAS CYTOCHROME c PEROXIDAS

IV THIOBACILLUS CYTOCHROME c PEROXIDASE (YAMANAKA, 1972)

V BACTERIAL CATALASES

VI GENERAL DISCUSSION

ACKNOWLEDGMENT

Chapter 16: OXYGENASES

Publisher Summary

I INTRODUCTION

II HISTORICAL BACKGROUND

III NOMENCLATURE AND CLASSIFICATION

IV NOHEME IRON-CONTAINING MONOOXYGENASES

V NONHEME IRON-CONTAINING DIOXYGENASES

VI HEME-CONTAINING OXYGENASES

VII CONCLUDING REMARKS

ACKNOWLEDGMENTS

Chapter 17: OTHER IRON-CONTAINING OR IRON-ACTIVATED ENZYMES: ENZYMES ACTING ON CERTAIN AMINO ACIDS, AMINES, AND ACETYL PHOSPHATE

Publisher Summary

I INTRODUCTION

II LYSINE 2,3-AMINOMUTASE

III L-SERINE DEHYDRATASE

IV SARCOSINE DEHYDROGENASE

V SPERMIDINE DEHYDROGENASE

VI PHOSPHOTRANSACETYLASE

PART IV: REACTIONS OF INORGANIC SUBSTRATES

Chapter 18: THE IRON-OXIDIZING BACTERIA

Publisher Summary

I INTRODUCTION

II CULTURAL CHARACTERISTICS

III IRON OXIDATION AND ENERGY PRODUCTION

IV INORGANIC SULFUR OXIDATION

V CARBON DIOXIDE FIXATION

VI HETEROTROPHIC METABOLISM

Chapter 19: MICROBIAL CORROSION OF IRON

Publisher Summary

I HISTORICAL BACKGROUND

II ECONOMIC SIGNIFICANCE

III PRINCIPLES OF CORROSION

IV MICROORGANISMS INVOLVED IN CORROSION OF IRON

V MECHANISMS OF MICROBIAL CORROSION

VI PREVENTION OF BIOLOGICAL CORROSION

PART V: MEDICINE AND CHEMOTHERAPY

Chapter 20: BACTERIAL IRON METABOLISM IN INFECTION AND IMMUNITY

Publisher Summary

I INTRODUCTION

II THE EFFECTS OF IRON AND IRON-BINDING PROTEINS ON BACTERIA IN VIVO AND IN VITRO

III CLINICAL ASPECTS OF ALTERED IRON METABOLISM AND INFECTION

IV THE INTERACTION BETWEEN BACTERIA AND IRON-BINDING PROTEINS

V THE EFFECT OF ANTIBODY AND IRON-BINDING PROTEINS ON BACTERIAL ETABOLISM

VI SUMMARY

AUTHOR INDEX

SUBJECT INDEX

CONTRIBUTORS

P.D. BRAGG

J.J. BULLEN

R.H. BURRIS

B.R. BYERS

JIANN-SHIN CHEN

ROGER A. CLEGG

CHARLES N. DAVID

THOMAS EMERY

E. GRIFFITHS

YUZURU ISHIMURA

WARREN P. IVERSON

NICHOLAS J. JACOBS

P. ANN LIGHT

WALTER LOVENBERG

D.G. LUNDGREN

RICHARD E. MILLER

LEONARD E. MORTENSON

J.B. NEILANDS

MITSUHIRO NOZAKI

K. OKUNUKI

W.H. ORME-JOHNSON

HENRY J. ROGERS

H. ROSENBERG

RICHARD D. SAGERS

STANLEY L. STREICHER

F.R. TABITA

RAYMOND C. VALENTINE

J.R. VESTAL

T. YAMANAKA

TAKASHI YONETANI

I.G. YOUNG

Copyright

COPYRIGHT © 1974, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED.

NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by

ACADEMIC PRESS, INC. (LONDON) LTD.

24/28 Oval Road, London NW1

Library of Congress Cataloging in Publication Data

Neilands, J B

Microbial iron metabolism: a comprehensive treatise.

Includes bibliographies.

1. Microbial metabolites. 2. Iron metabolism.

I. Title.

QR88.N44 576′.11′33 73-18956

ISBN 0-12-515250-7

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS

Numbers in parentheses indicate the pages on which authors’ contributions begin.

P.D. BRAGG(303),     Department of Biochemistry, University of British Columbia, Vancouver, British Columbia

J.J. BULLEN(517),     National Institute for Medical Research, Mill Hill, London, England

R.H. BURRIS(187),     Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin

B.R. BYERS(83),     Department of Microbiology, University of Mississippi School of Medicine, Jackson, Mississippi

Jiann-Shin CHEN(231),     Department of Biological Sciences, Purdue University, West Lafayette, Indiana

ROGER A. CLEGG(35),     Department of Biochemistry, Medical Sciences Institute, The University, Dundee, Scotland

CHARLES N. DAVID*(149),     Division of Biology, California Institute of Technology, Pasadena, California

THOMAS EMERY(107),     Department of Chemistry and Biochemistry, Utah State University, Logan, Utah

E. GRIFFITHS(517),     National Institute for Medical Research, Mill Hill, London, England

YUZURU ISHIMURA(417),     Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

WARREN P. IVERSON(475),     National Bureau of Standards, Washington, D.C.

NICHOLAS J. JACOBS(125),     Department of Microbiology, Dartmouth University Medical School, Hanover, New Hampshire

P. ANN LIGHT(35),     Department of Radiotherapy, Research Floor, Outpatients Building, Bristol Royal Infirmary, Bristol, England

WALTER LOVENBERG(161),     Section on Biochemical Pharmacology, Experimental Therapeutics Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland

D.G. LUNDGREN(457),     Department of Biology, Biological Research Labs, Syracuse, New York

RICHARD E. MILLER*(283),     Laboratory of Biochemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland

LEONARD E. MORTENSON(231),     Department of Biological Sciences, Purdue University, Lafayette, Indiana

J.B. NEILANDS(3),     Department of Biochemistry, University of California, Berkeley, California

MITSUHIRO NOZAKI(417),     Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

K. OKUNIKI(349),     Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan

W.H. ORME-JOHNSON(187),     Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin

HENRY J. ROGERS(517),     National Institute for Medical Research, Mill Hill, London, England

H. ROSENBERG(67),     Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, Australia

RICHARD D. SAGERS(445),     Department of Microbiology, Brigham Young University, Provo, Utah

STANLEY L. STREICHER(211),     Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

F.R. TABITA†(457),     Department of Chemistry, Washington State University, Pullman, Washington

RAYMOND C. VALENTINE(211),     Department of Chemistry, University of California at San Diego, La Jolla, California

J.R. VESTAL(457),     Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

T. YAMANAKA(349),     Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan

TAKASHI YONETANI(401),     Johnson Research Foundation, Department of Biophysics and Physical Chemistry, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

I.G. YOUNG(67),     Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, Australia

*Present Address: Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York

*Present Address: Division of Metabolic Disease, Department of Medicine, University of California at San Diego, La Jolla, California 92037

†Present Address: Department of Microbiology, University of Texas at Austin, Austin, Texas 78712.

PREFACE

The rich diversity of genetic material in microorganisms renders them ideal subjects in which to study the metabolism of any substance essential for life processes. Their short generation time, the plasticity of their metabolism, and the ease of handling of microbial cultures are additional reasons for selecting the unicellular forms of life for the studies reported in this volume. Yet the certain unity of cells through the microbial, plant, and animal worlds suggests that fundamental discoveries in one may be applied to all.

Although iron is one of the most commonly encountered elements on earth and throughout the solar system, its concentration in living cells is characteristically low, and hence it is rightly classed as a trace element. The amount of iron in the human body is 4 to 5 grams, which is the weight of a small nail. But the element is involved in biological reactions that are crucial to life, among which are the transfer of electrons at redox potentials below (ferredoxins) and above (cytochromes) the pyridine nucleotides, the transport of oxygen, the reduction of O2 and N2, the reduction of ribotides to deoxyribotides for the synthesis of DNA, and the metabolism of inorganic nitrogen compounds.

A truly comprehensive treatment of microbial iron metabolism would be difficult to compress within one small volume. Similarly, the definition microbial has not been extended to algae and protozoa. In spite of these limitations it is hoped that this book will speed the total understanding of the path of iron in microbial species and, eventually, in the plant and animal. This fundamental knowledge can be used for good or evil. It is the special responsibility of the scientist to guard against the perversion of basic information and to strive for the humanization of our profession. I believe that this should be our most important priority for the remainder of this century.

I take this opportunity to thank the contributors for their care in the preparation of the manuscripts and for their courtesy in meeting deadlines. The staff of Academic Press has been cooperative at all stages in the evolution of this work.

J.B. Neilands

PART I

INTRODUCTION

Outline

Chapter 1: IRON AND ITS ROLE IN MICROBIAL PHYSIOLOGY

Chapter 2: METABOLISM IN IRON-LIMITED GROWTH

Chapter 1

IRON AND ITS ROLE IN MICROBIAL PHYSIOLOGY

J.B. NEILANDS

Publisher Summary

This chapter describes iron and its role in microbial physiology. A prime function of iron in aerobic microbial species is in respiration—the reduction of oxygen by means of the cytochrome chain and the concomitant generation of chemical energy. A resume of iron metabolism in microbes, plants, and animals shows some common features, but it mainly reveals how much remains to be done in this field. Aerobic and facultative aerobic microorganisms synthesize siderochromes, the biofunction of which has been confirmed by the techniques of biochemical genetics to be iron transport. Iron carrier molecules are also suspected to occur in plants and animals. The siderochromes are, with one exception, highly polar and water soluble. Storage forms of iron have been documented thoroughly in animals, less well in plants, and only poorly in microorganisms.

I Introduction

II Historical Background

A Experiments of O. Warburg

B Experiments of D. Keilin

III Biogeochemistry of Iron

A Distribution

B Evolutionary Transformations

IV Some Physical and Chemical Properties of Iron

A Inorganic Chemistry

B Coordination Chemistry

C Biochemical and Biophysical Techniques

V Iron Content of Microorganisms

VI Iron Ligand Atoms in Microorganisms and Their Function

A All-Oxygen

B Mixed Oxygen-Nitrogen

C Mixed Oxygen-Sulfur

D All-Nitrogen

E Mixed Nitrogen-Sulfur

F All-Sulfur

VII Life without Iron and Functional Replacements for Iron

A Lactobacilli

B Flavodoxin

VIII Aspects of the Comparative Biochemistry of Iron Metabolism

A Plants

B Animals

C Comparison of Species

References

I INTRODUCTION

This chapter is intended to cover some assorted features of iron and the function of this element in the microbial world. Doubtless it will reflect a conspicuous prejudice of the author, namely, that an appreciation of structure is fundamental to an understanding of biological performance.

Included in this chapter are, among other topics, a historical sketch of the recognition of the importance of iron in cellular physiology, a description of certain physical properties of ferrous and ferric ions, and a list of various known biocoordination derivatives grouped by ligand atom. Obviously, no single aspect of this conglomerate can be treated in detail. However, it is hoped that the appended references will provide at least an entrée to the literature.

Some iron enzymes—aconitase and succinic dehydrogenase come to mind—although highly purified from animal sources, have as yet been somewhat less thoroughly investigated in microorganisms. The tacit assumption that the microbial counterparts of mammalian iron enzymes will contain or require this metal needs to be verified experimentally in view of the known ability of the proton to substitute for metal ions. Conversely, we need to be aware that microbes may possess enzymes which are uniquely iron-containing.

Microbial iron metabolism, like the field of antibiotic research, has been plagued by problems of nomenclature. Iron transport compounds have been described, by various authors, as siderochromes, ferric ionophores, ironophores, and ferriphores (Hutner, 1972). A future session of a Gordon Conference, or perhaps a more international body, should be commissioned to devise a systematic name which will integrate the nomenclature for all other carriers which transport substances across the microbial membrane.

II HISTORICAL BACKGROUND

In microbes, as in most other living tissues, the bulk of the iron present can be described generally as catalytic, or enzyme, iron which is engaged in electron transport processes of one sort or another (Coughlan, 1971). A catalytic role is true even for hemoglobin iron if atmospheric oxygen is regarded as substrate and tissue oxygen the product.

A prime function of iron in aerobic microbial species is in respiration—the reduction of oxygen by means of the cytochrome chain and the concomitant generation of chemical energy. However, it has been known for some time that anaerobic species may also contain substantial quantities of iron. Certain members of the Clostridia, in particular, are a rich source of the iron-sulfur proteins.

The importance of iron in cell physiology has been revealed by the work of a large number of investigators, preeminent among them Otto Warburg (1883–1970) and David Keilin (1887–1963). Fortunately, in addition to their monumental research, each has left a personal statement containing a complete and often pithy account of their work and ideas (Warburg, 1949; Keilin, 1966).

Their discoveries promoted iron from a limited status (O2 transport) to a full partnership (respiration) in life processes.

A Experiments of O. Warburg

Warburg studied the oxidation of various organic compounds by blood charcoal and noted the sensitivity of these model systems to inhibition by cyanide. He concluded that … iron combined with nitrogen produced the chemically active regions…. These experiments were extended to sulfhydryl compounds in solution. Ultimately, he demonstrated that cyanide and carbon monoxide poison the ferric and ferrous forms, respectively, of the Atmungsferment. The latter was characterized as a heme compound by measurement of the photochemical absorption spectrum of its carbon monoxide complex (Warburg, 1949).

B Experiments of D. Keilin

Keilin (1966) rediscovered the pigments myohematin and histohematin which were observed in echinoderms to man a half-century earlier by MacMunn. Keilin, armed mainly with a low-dispersion microscope, reasserted MacMunn’s belief that the tissue pigments functioned in cellular respiration and he named them cytochromes. In his earliest papers on the subject, Keilin stressed the ease of detection of the cytochromes in the cells of aerobic bacteria and yeast. He is thus credited with the discovery of the central role of iron in respirations (Malmström, 1970).

III BIOGEOCHEMISTRY OF IRON

A Distribution

In the solar system, the general exponential decrease in abundance of nuclides in the progression of mass numbers up to about 100 is interrupted by a sharp peak centered at mass 56 (Burbridge et al., 1957). This is attributed to the favored formation of the stable ⁵⁶Fe nucleus in the complicated series of fusion reactions taking place at high temperatures in the stars. Iron is believed to be present in interstellar dust in significant amounts (Chaisson, 1972) and it comprises about 40% of the weight of certain meteorites (Zajic, 1969).

The ocean, which covers fully 70% of the surface of Earth, contains about 0.01 ppm iron, which corresponds to 0.2 × 10−6 gram atoms per liter (Bowen, 1966), the concentration increasing with depth. The fertility of the ocean may depend to some extent on upwelling currents which bring to the surface iron, the latter a rate-limiting nutrient for the growth of plankton and hence all marine life. River water is typically higher in iron, the concentration ranging between 0.01 and 1.4 ppm (Bowen, 1966).

The whole planet Earth contains about one-third iron, the concentration being higher than that of any other single element. On the Earth’s crust it is over-shadowed somewhat by silicon and oxygen, but even here only aluminum outranks iron in abundance among the metals (Zajic, 1969). Igneous rocks, shale, sandstone, and limestone may contain 5.6, 4.7, 1.0, and 0.4% iron, respectively. The mean concentration in soil is 3.8% (Bowen, 1966). The availability of soil iron to plants depends on a multiplicity of factors, among which are pH and microbial action.

B Evolutionary Transformations

Iron, as well as all the other elements, is known to have been present in the reduced state on the surface of the primitive Earth (Ponnamperuma, 1971). The fossil record suggests that blue-green algae could have been present several billion years ago (Oehler et al., 1972). Assuming the age of the earth as 4.5 × 10⁹years, there was possibly a billion years of chemical, organic, and biological evolution throught the Clostridia -S-S- couples.

Zajic (1969) has prepared a list of iron minerals, ranging from siderite (FeCO3) to ferromanganese concretions, and has speculated as to which microbial species may have beeninvolved in their deposition or dissolution. A most comprehensive description of the ability of microorganisms—bacteria, fungi, algae, and protozoa—to transform minerals on a geological scale has been recorded by Silverman and Ehrlich (1964).

Arrieta and Grez (1971) isolated a number of soil organisms, selected on the basis of their ability to solubilize the iron of granodiorite, and examined the capacity of ten pure fungal strains to dissolve six iron-containing minerals in the course of 21-day culture. In the case of two minerals with the same crystalline form, augite and biotite, the amount of iron liberated correlated with its abundance. However, both hematite (trigonal) and magnetite (cubic), although over 65% in iron, were dissolved to only a very slight extent. Aspergillus niger was studied in greater detail and the solubilizing action was ascribed to metabolic products of the fungus. Arrieta and Grez (1971) concluded that complex formation could be the mechanism of solubilization inasmuch as the culture iron displayed altered behavior on thin-layer chromatography and could not be removed from solution by cation-exchange resins. The Chilean workers were apparently unaware that A. niger synthesizes ferrichrome-type siderochromes (Garibaldi and Neilands, 1956).

Six lichen compounds, all of them sparingly soluble in water and most of them of unknown metal complexing ability, were shown to promote chemical weathering of various silicates, although control organic acids, such as citric, dissolved more iron and aluminum (Iskandar and Syers, 1972).

A systematic study of the ability of deferrisiderochromes to extract iron from minerals would be of considerable interest. These fungal products may have binding constants for ferric ion in excess of 10³⁰ (Anderegg et al., 1963) and they also show high affinity for a few other metal ions, such as aluminum. Laterite would be a particularly favorable substance for investigation since it contains a high proportion of oxides and hydroxides of iron and aluminum. Laterization is a significant problem in tropical soils, especially those which have been stripped of plant life through natural or human causes. The process is regarded as irreversible and soil once laterized is incapable of sustaining vegetative life. This may be one of the ultimate consequences of the military defoliation and craterization of Indochina (Galston, 1972).

The relationship of iron to the other elements important in biology is well illustrated by reference to Shaw’s elaborate biogeochemical table (Zajic, 1969), which lists the distribution as well as the chemical, physical, electronic, and other properties of the bioelements. Iron, together with the other transition elements, is placed in Division II, characterized by long runs of biologically inert elements. In general, such classifications can rationalize why only certain of the elements have been accepted or rejected by living cells. Thus all attempts to set up nutrient lines and bioloops in the Periodic Table cannot explain why some elements have not been called upon to perform a biofunction. As the nutritional sciences become more refined and quantitative some new elements can be expected to be discovered as essential for the living organism, including higher animals (Underwood, 1971).

IV SOME PHYSICAL AND CHEMICAL PROPERTIES OF IRON

A Inorganic Chemistry

1 Solubility

Pure iron wire which has been protected from the atmosphere is a satisfactory primary standard for iron determinations. Metallic iron dissolves easily in aqueous hydrochloric acid to give a solution of FeCl2 · 6H2O, which is pale green. On titration of a ferrous salt to neutral pH about one-half of the metal ion is precipitated as an hydroxide. The latter soon darkens in air and on further standing forms Fe2O3 · nH2O. The solubility product constant of ferrous hydroxide at 25°C and zero ionic strength is 10−15.1 (Leussing and Kolthoff, 1953).

The ferric ion is partially hydrolyzed even at pH values as low as zero. Treatment of a ferric ion solution with base will afford a precipitate which is one-half formed at a pH of about 3. Contrary to popular notion, ferric hydroxide does not have the precise constitution Fe(OH)3 but consists, rather, of various polynuclear structures. The latter have been discussed by Spiro and Saltman (1969). The solubility product constant for ferric hydroxide has been estimated by Kolthoff and Elving (1962) as 1.1 × 10−36 and as 10−38.7 by Biedermann and Schindler (1957). The binding affinity for the perchlorate anion to ferrous and ferric (or other metal) ions is close to nil and for this reason perchlorate salts are the most useful for all types of quantitative complexometric investigations. The very low solubility of free ferrous and ferric iron at physiological pH suggests that these ions will more commonly occur in prosthetic groups and tightly bound to proteins, and that they will have a less conspicuous role as activators. This low solubility also provides a prime raison d’être for the siderochromes.

2 Oxidation Reduction Potential

Iron, the most versatile of all the biocatalytic elements, owes its wide spectrum of chemical reactivities to its two stable valencies and to the considerable range of oxidation reduction potential connecting this coupe. This is the property which enables the efficient trapping of three quanta of energy, amounting to about 8 kcal each, per oxygen atom reduced by the electron transport chain. Similarly, the substantial latitude observed in the generally lower potential of the iron-sulfur proteins can account for the many important enzyme reactions of these substances. Clearly, the factors which cause the standard reduction potential of iron to span the scale from +300 mV in a-type cytochromes to −500 mV in certain iron-sulfur proteins are those which endow iron with its amazing capacity to engage in electron transport reactions.

The factors affecting the potential of heme compounds have been the subject of much discussion and experimentation (Falk, 1964). The insertion of electron-withdrawing substituents at the β positions of the pyrrole nucleic will tend to raise the potential to some extent. Thus the potential, at pH 9.6, of −63 mV for mesoheme rises to +4 mV in hematoheme and to +15 mV in protoheme as the saturated porphyrin side chains are substituted successively with hydroxyl and vinyl groups (Falk, 1964). Further perturbations of the potential have been demonstrated in hemes bearing various nitrogen bases as the axial ligands. Again, however, the effect is relatively small and it cannot account for the latitude of redox potentials noted in the various cytochromes.

Recently it has been suggested that the degree of hydrophobicity of the heme milieu may be the most important factor in determining the potential. The problem has been to find a model in which the heme potential could be raised to the range observed experimentally for the cytochromes. Kassner (1972) showed that there is a 300 mV elevation in potential between the dipyridine hemochromogen of mesoheme 1X in aqueous solution and the ester of the same compound in benzene solution. Dickerson et al. (1972) cite a number of other cases, all of which support the theory of hydrophobicity as the main determinant in establishing the particular value of the potential. Thus the heme-bearing fragments of cytochrome c studied by Harbury et al. (1965), the heme c peptides, have potentials just about 300 mV below that of cytochrome c. Dickerson’s X-ray structures at < 3 Å for both oxidized and reduced cytochrome c show the heme group lying in a cavity of low dielectric constant. Two other cytochromes have been analyzed by X-ray diffraction, cytochrome b5 and the c2 of Rhodospirillum. These show the heme situated in a less and more hydrophobic environment, respectively, and —as expected —with reference to cytochrome c the potentials are in turn lower and higher. Mitochondrial cytochrome b has a potential of +35 mV in situ and −340 mV in aqueous solution.

A 4-iron 4-sulfur protein from Chromatium, HiPIP, is unique among the iron–sulfur proteins in that it has a redox potential of +350 mV. Characteristically, the redox potentials of the iron-sulfur proteins are low and fall within the range −230 to −430 mV (Tsibris and Woody, 1970). HiPIP and the 8-iron 8-sulfur ferredoxins appear to contain an identical Fe4S4 cluster, which can be regarded as an inorganic cubane. Carter et al. (1972) have proposed that the Fe4S4 center has three oxidation states; this would resolve the paradox of the similar structure but the widely different redox potentials seen in the ferredoxins and HiPIP. Thus reduced HiPIP would correspond to oxidized ferredoxin and super reduced HiPIP would be equivalent to reduced ferredoxin.

B Coordination Chemistry

1 Ligand Field

In essence, the ligand field theory of coordination places the central metal ion in an environment where the binding atoms surrounding it may affect certain of the metal electrons to a variable degree (Phillips and Williams, 1966). As a transition element, iron possesses unfilled d orbitals and these may be differentiated by coordination into lower and higher energy levels than those existing in the uncomplexed ion. Ligands designated strong field tend to force the d electrons into spin-paired orbitals while those described as weak field allow the d electrons to assume the configuration of the free ion, i.e., maximum unpaired parallel spins. The limits for the two types of ligands impinging on both ferric and ferrous ions are shown in Fig. 1. Obviously, knowledge of the field strength of the ligands present in a system, coupled with quantitative measurements of the paramagnetic susceptibility in both oxidation states, can provide important clues regarding the nature of the atoms linked to the metal ion. Water and halide ions tend to be weak field, while cyanide and ammonia are classed as strong field. The relation between spin state and structure can be illustrated by reference to a series of hemoglobin derivatives in which the metal, whether ferric or ferrous, is always bonded to four pyrrole nitrogens and one imidazole nitrogen. When the sixth ligand is water, as in deoxyhemoglobin, the ferrous ion has four unpaired electrons and is high spin. In the methemoglobin series, where iron is ferric, the fluoride and cyanide complexes have five and one unpaired electrons and are, respectively, high and low spin. Cytochrome c, whether oxidized or reduced, is always low spin because the sulfur of methionine residue No. 80, a strong field ligand, remains in the coordination sphere of the iron (Wüthrich, 1970).

Fig. 1 Ferric and ferrous ions in weak and strong ligand fields.

Martin and White (1968) have reviewed specifically the nature of the transition between low- and high-spin states in octadehral complexes.

2 Stability and Structure

The stepwise association of ligands with ferric or ferrous ions results in the formation of coordination derivatives which are often octahedral, occasionally tetrahedral and sometimes, especially in biological systems, of irregular geometrical configuration in solution. The chelate effect generally affords some additional stability. Thus the log formation constant for ferric triacethydroxamate is 28.3 while the corresponding value for the ferrichromes, which are cyclic hexapeptides containing a set of three coordinating hydroxamates per mole, are one to four orders of magnitude higher (Neilands, 1966). However, for maximum positive effect the ligand groups must not be sterically constrained from clustering around the central metal ion, otherwise the effect is liable to be negative. Like the other metal ions, ferrous and ferric ions prefer to be chelated in five- or six-membered rings, the former ring size providing the most favored angles and, hence, the more stable compounds except in special ligands such as the porphyrins. Although stability constants have not been published for the iron porphyrins, an estimate of relative stabilities can be obtained from replacement reactions (Falk, 1964). The iron goes in and out best in the ferrous state, but in vitro both reactions require energetic conditions relative to the enzymatic insertion. Proteins, because of their tertiary structure, can act as ligands with trans spanning and may have very large rings. In summary, the stability of the iron chelate will depend on a multitude of parameters such as ring size, resonance effects, nature and basicity of the donor atoms, the magnitude of the chelate effect and, finally, steric and conformational factors. The standard methods for measuring stability constants have been summarized by Rosotti and Rosotti (1961).

In its propensity to form chelates with a given ligand, ferric and ferrous ions are somewhat intermediate in activity among the transition elements. Both ions possess a relatively small size, a high charge, and a number of d orbitals available for bonding. Ferric ion displays a distinct preference for oxygen ligands and forms stable complexes (chelates) with β-diketones, catechols, and hydroxamates. Ferrous ion, on the other hand, binds more tightly to all-nitrogen ligand systems such as the 1,10-phenanthrolines (Phillips and Williams, 1966). Obviously, the metalloporphyrins and all other natural chelates which function by reversible oxidation and reduction must have a molecular architecture which efficiently binds both valence states of the coordinated ion.

In reconstitution experiments it is often expeditious to add back the iron in its more soluble ferrous form and, if desired, to allow the oxidation to occur in situ.

Ferric compounds of EDTA and deferriferrichrome-type compounds exchange their iron with half-times of minutes or hours, depending on conditions, but with the porphyrins the rate is infinitely slow. Iron can in principle be removed from all coordination states with acid, alkali, or by treatment with more powerful chelators. The latter are commercially available in both hydrophilic and hydrophobic forms, the former being especially useful for metalloproteins. Chelex, a polyaminocarboxylic acid resin, has been applied for both cleaning media and for removing iron from proteins.

C Biochemical and Biophysical Techniques

1 Coordination Reagents

a Inhibitors

The classical inhibitors for iron enzymes have been cyanide and carbon monoxide, especially the former (Keilin, 1966). The cyanide ion is a particularly versatile ligand and it even outranks halides in its binding ability. It acts as a general scavenger for heavy metals, but it is well to recall that it also binds to carbonyl groups and to pyridine nucleotides. If cyanide owed its primary poisonous property to attachment to hemoglobin iron then many grams would be required to kill a human whereas, in fact, the mortal dose is probably of the order of millimoles. Cyanide thus binds to ferricytochrome oxidase and effects an asphyxia at the tissue, rather than at the circulatory, level. Amyl nitrite is an effective antidote for cyanide intoxication, if used promptly, since it converts some of the hemoglobin to methemoglobin which displays, like all ferric porphyrins, a superior avidity for cyanide. Carbon monoxide, on the other hand, reacts preferentially with the ferrous porphyrin since the divalent iron has a greater tendency to donate electrons to the unfilled π orbitals of the ligand. In the case of cytochrome oxidase, Warburg (1949) was able to regenerate active enzyme by illuminating the complex with those wavelengths of light which were absorbed by the heme nucleus of the enzyme.

No coordinating agent will be found absolutely specific for any single metal ion. The siderochromes, which have binding constants of 10³⁰ or greater for ferric ion (Anderegg et al., 1963), are relatively specific for this trivalent ion. The stability constant for the 3:1 complex of 8-hydroxyquinoline with ferric ion is greater, i.e., 10³⁸ (Sillen and Martell, 1964), but in this case the ligand binds a number of metals, including ferrous ion, with substantial strength. Bragg (see Chapter 13) has described the application of a number of chelating agents for investigation of the nature of the nonheme iron of the respiratory chain.

b Chromogenic Reagents

The most popular reagents for colorimetric determination of iron in biology, where the concentration may often be less than 10−5 gram atoms per liter, are the ferroins, i.e., substituted dipyridyls of phenanthrolines (Carter, 1971). Bathophenanthroline (4,7-diphenyl-1,10-phenanthroline) and 2,4,6-tripyridyl-1,3,5-triazine have molar absorptivities with ferrous ion of 22,150 and 22,600 at 534 and 593 nm, respectively. The most potent chromogen of the series is terosite, a terpyridine (Zak et al., 1971). The strong absorptivities at longer wavelengths are responsible for the brilliant magenta colors of the ferrous coordination compounds, which enable accurate determination of somewhat less than 1 µg of iron. Generally, combustion of the sample is necessary in order to liberate the iron and this means that the analysis must be buffered before the spectrophotometric step. A considerable degree of contamination can be introduced in this way since acetate (Van de Bogart and Beinert, 1967) and phosphate (Nelson, 1964) salts contain substantial amounts of iron.

An experimental hazard which needs to be appreciated in all attempts to define the oxidation state through use of the ferroins is that the analysis will always tend to drift positive for ferrous iron. This is because in the presence of any organic matter or other source of electrons, particularly sulfhydryl groups, the ferrous iron will be stabilized and will accumulate as a consequence of its stronger binding to the ligand.

Substituted ferroins are routinely used for distinguishing cuprous and cupric copper (Komai and Neilands, 1969). Thus, a 2,9-dimethylphenanthroline (a cuproine) is sterically confined to complexation with the tetrahedral cuprous ion while cuprizone (biscyclohexanone oxaldihydrazone) is selective for the square planar cupric ion.

The classical method for determination of heme iron involves first splitting the conjugated proteins with acid acetone followed by determination of the heme as its hemochromogen with pyridine (Falk, 1964). The nonheme iron is given as the difference between total iron and heme iron (see Chapter 13).

2 Direct Total Iron Determination

The most versatile and generally applicable method of iron determination in microbiology is atomic absorption spectroscopy (Ambrose, 1971), first described by Walsh (1955). Neutron activation analysis is capable of yielding equally reliable results but it is encumbered by the need for a neutron source. Atomic absorption has the profound advantage of obviating the prior combustion step which is nearly always mandatory in colorimetric analyses. Stripped to its simplest description, the atomic absorption spectrometer consists of a hollow cathode discharge tube, a heat supply capable of presenting the sample as a cloud of atoms, a monochromator and, finally, a detector. The emission source must be chopped or otherwise modulated to screen out line spectra emission in the flame. With this type of arrangement, the now classical flame-type atomic absorption spectrometer, it is possible to obtain satisfactory analysis on approximately 0.5 ml of solution containing about 10−5 gram atoms of iron per liter, or about 0.25 µg iron. This is about as sensitive as the colorimetric methods employing a reagent such as bathophenanthroline, which has a molar absorbancy of 22,150 at 534 nm (Carter, 1971).

Since most of the sample is wasted by being continuously swept into the flame over the period of time required to reach a stable reading, much effort has been expended to perfect a more economical means of presenting the sample. Small sampling boats can be used to increase the sensitivity for some metals, but not for iron. The modern approach is to turn to completely flameless atomization, an adaptation which usually augments the sensitivity by several orders of magnitude (Robinson and Slevin, 1972). With this type of apparatus it is possible to measure the lead content of a 1-cm section from a single strand of human hair (Renshaw et al., 1972). In the Perkin-Elmer HGA-2000 furnace a sample up to 100 µl is pipetted through a hole in a 50 × 10 cm cylinder mounted horizontally. A three-stage, automatically programmed current is passed through the cylinder walls which successively dries, ashes, and atomizes the sample. According to Amos (1972) and Kahn (1972) this type of furnace has an absolute detection limit for iron of 50 and 3 pg, respectively. A further advantage of flameless atomic absorption is that it can be applied to liquid and solid samples.

Atomic absorption will never displace entirely the use of chelating agents. The latter are cheap, they can be employed as inhibitors and as a means of detecting the valence state, they are often required for exchanging a metal ion or introducing an isotope and, finally, they can be used for extracting contaminating metal ions from culture media.

3 Radioactive Iron

Radioactive iron affords a means of achieving utmost sensitivity of measurement and, provided the metal ion is not rapidly exchanged, it gives a means of tracing iron coordination compounds through metabolism. Radioiron is also useful in all types of experiments involving ferrokinetics, intracellular pools, and radioautography, although these techniques have as yet been applied on a very limited scale in microorganisms. Two unstable isotopes of iron are widely used, ⁵⁵Fe and ⁵⁹Fe, and both are available commercially in a variety of salts and citrate complexes (Sommerville, 1967). Although readily counted, ⁵⁹Fe has a half-life of only 45 days—which can be an advantage in some cases. Iron-55 decays by electron capture and has a half-life of 2.94 years. Siderochromes, such as Desferal, have been proposed as a means of coordinating ⁵⁵Fe to increase counting efficiency in scintillation fluids (Haydon et al., 1972).

4 Physical Probes

The physical probes, including optical rotatory dispersion and circular dichroism, applicable to metalloproteins have been reviewed by Vallee and Wacker (1970). In contrast to some of the other biometals, iron affords a rich variety of signals for biophysical experimentation.

a Electronic Spectra

The spectra of iron porphyrins have been discussed by Falk (1964). A heme compound is readily detected via its Soret band at about 400 nm, which characteristically has a molar absorbency in excess of 10⁵. The brilliant red colors of the hemochromes, which are hemes with further nitrogenous ligands in both axial positions, can be attributed to the α and β bands lying between about 500 and 600 nm. The spectra of ferricytochrome c and ferrocytochrome c are shown in Fig. 2 (1) and (2), which illustrates the merit of performing light absorption measurements with the reduced form of the iron porphyrins. Obviously, ferrocytochrome c displays a hemochrome-type spectrum without the addition of further ligands since in this case the iron is attached to histidine No. 18 and the sulfur of methionine No. 80.

Fig. 2 Absorption spectra of some iron compounds from microbial sources. (1) Ferrocytochrome c; (2) ferricytochrome c; (3) upper curve: ferric trihydroxamate, pH 7; lower curve: ferric monohydroxamate, pH 2; (4) a microbial ferredoxin.

The ferric hydroxamates exhibit pH dependent bathochromic and hipsochromic shifts in their visible absorbancy. The 3:1 complexes, which are favored at neutral pH, are typically reddish tea colored and have a broad maximum centered at about 430 nm [Fig. 2 (3)]. The natural polyhydroxamates largely retain this spectrum at low pH but the monohydroxamates compete less favorably with acid and at pH 2 ferric triacethydroxamate is degraded to the 1:1 form. The latter is purple in color, the maximum is at around 500 nm, and the intensity of absorbency drops to a third of the value at neutral pH.

The ferredoxins have general absorption, without sharp peaks, through the lower visible region of the spectrum and are brownish in color [Fig. 2 (4); Lovenberg, this volume, Chapter 8).

The ligand field theory enables certain predictions regarding the electronic spectra of coordination compounds (Phillips and Williams, 1966). Gray (1971) has demonstrated the value of spectral analysis for elucidation of the nature of the iron-binding center in hemerythrin. Many of the brilliantly colored metal compounds encountered in biology are believed to be the result of charge transfer, either M→L or L→M.

b Magnetic Susceptibility

It has been already noted that, as a transition element, iron may have unpaired electrons in the d-shell orbitals and thus display paramagnetism. Quantitative determination of the number of unpaired electrons through measurements of magnetic susceptibility can afford information on the metal ion valence and its bond type in coordination states (Figgis and Lewis, 1960). This method has traditionally been of limited value for iron proteins because of the difficulty in obtaining sufficient sample. Hemoglobin is an exception, of course, and the early observation of Pauling and Coryell (1936) that the deoxy form is high spin with four unpaired electrons while the oxy form is low spin with zero unpaired electrons has enabled speculations regarding the trigger mechanism for cooperativity among subunits in the tetramer. Thus a movement of the iron in and out of the porphyrin plane, which is accompanied by a change in spin state, during oxygenation and deoxygenation may set in motion conformational changes in the polypeptide chains (Hoard, 1971; Perutz and Ten Eyck, 1971). An ultrasensitive magnetometer, designed on the basis of superconductivity, has been applied for investigation of the environment of hemerythrin iron, which is thought to be oxobridged (Dawson et al., 1972).

c Electron Spin Resonance

Since its introduction in 1945, the techniques of ESR has been applied to two general classes of paramagnetic substances, the transition ions and the organic free radicals. Synthetic, stable organic free radicals (spin labels) have proved useful for the study of membranes and biological processes. The reader is referred to the publications of McConnell, who has pioneered this field (McConnell and McFarland, 1970). The theory of ESR and its application to biological systems have been dealt with in a number of publications (Pake, 1962; Ehrenberg et al., 1967; Wyard, 1969; Swartz et al., 1972).

As a probe for paramagnetism, ESR is somewhat more convenient than magnetochemistry and for this reason it has been used extensively for structural studies of iron proteins, especially the ferrodoxins (Lovenberg, this volume Chapter 8). The latter display an unusual and highly characteristic signal at g = 1.94. Replacement of the ⁵⁶Fe by ⁵⁷Fe and the ³²S by ³³S in some of these proteins followed by monitoring of the changes in the ESR spectra confirms the iron-sulfur bonding. An analysis of ESR spectra at various temperatures may be employed to identify ligands attached to ferric ion in a completely rhombic field (Peisach et al., 1971).

d Mössbauer Spectra

Nuclear gamma-ray resonance spectroscopy is possible in only certain isotopes, one of which is ⁵⁷Fe (Greenwood and Gibb, 1971). The technique is useful for collecting data on the equivalence or nonequivalence of the ferrous/ferric ions when several of these are present per mole, as in the ferredoxins. Bearden and Dunham (1970) have summarized the information available from Mössbauer spectroscopic studies of iron proteins; the heme proteins are relatively well understood (Lang, 1970).

According to a Mössbauer investigation of protocatechuic acid 4,5-oxygenase the iron atoms may be low-spin ferrous, or the enzyme may contain two antiferromagnetically coupled high-spin ferric ions. The latter interpretation was favored (Zabinski et al., 1972). The symmetrical coordination propeller characteristic of the siderochromes is incompatable with their Mössbauer spectra and their ESR signal at g = 4.3, which are typical of high spin FeIII in rhombic field. This problem is being investigated by W. T. Oosterhuis and his colleagues at Carnegie-Mellon University.

V IRON CONTENT OF MICROORGANISMS

Stephenson (1949), in her classical work on bacterial metabolism, cites ten species which had iron contents ranging from 0.0036 to 0.0175% on a dry weight basis after growth as surface cultures on peptone agar. This averages to about 0.01% dry weight, or 0.1 mg/gm.

Warburg (1927) early dispelled any expectation that only aerobic organisms should contain high levels of iron. He grew Torula utilis aerobically and another yeast, Strain U, anaerobically and then measured the iron content of the cells. Both contained about 0.1 mg/gm dry wieght. He estimated that most cells should fall within the range 0.01 to 0.1 mg/gm. Elvehjem (1931), in a now classic study of the iron nutrition of yeast, reported up to 24 mg/gm dry weight of cells. Curran et al. (1943) showed the iron content of Clostridium sporogenes to be over four times that of Bacillus subtilis. The iron-sulfur proteins may contain several percent iron whereas in the cytochrome series even the smallest member, component c, contains only 0.45% of the metal. Of course, what counts biologically is the catalytic activity per gram atom of iron.

Unfortunately, a systematic survey of the iron level of the main representatives of the microbial world seems not to have been carried out at this writing. Given the modern instrumentation, such as atomic absorption spectroscopy, this is no longer a formidable task. Substantial differences can be anticipated, of course, which depend merely on the environmental and cultural conditions. Presented with a sugar substrate and no added iron, a facultative cell might be expected to derive energy mainly from glycolysis and the cells would be low in cytochromes. Growth on a TCA cycle intermediate would force the cell to augment its iron content if its only means of obtaining energy were via oxidations through the cytochrome chain.

In regard to the iron content of different types of microorganisms, Bowen (1966) quotes references citing levels of 3.5, 0.25, and 0.13 mg/gm dry weight as the iron content in plankton, bacteria, and fungi, respectively. The corresponding figures quoted for copper were 0.2, 0.042, and 0.015 mg/gm. This suggests that copper can be expected to occur at a level about one order of magnitude below that of iron. Hutner (1972) has published a scholarly review of the inorganic nutrients of microorganisms, with special emphasis on iron and methodology; the earlier literature has been reveiwed by Knight (1951).

While all living cells have the capacity to concentrate iron, the species with the most extraordinary ability to perform this act are the ocean plankton. Of the trivalent ions, iron is the most strongly concentrated (Bowen, 1966). The concentration factor for plankton and brown algae may be as high as 100,000, but the biochemical mechanism for this effect has not been elucidated (Spencer et al., 1972).

How much iron should be added to culture media? Waring and Werkman (1942) worked out extraction methods for lowering the iron contamination of media. They determined that the common enteric bacteria needed 0.02–0.03 mg/liter iron for maximum growth while Pseudomonas aeruginosa required the element at a fourfold higher level. Even a very aerobic species will not give a packed cell yield of over 10% v/v. Assuming the packed cells to be 80% water and assuming further that the cells contain 0.1 mg/gm dry weight of iron, each liter of medium would have to be supplied with 2 mg of iron. Allowing for inefficient utilization, 10 mg of added iron per liter should more than suffice to meet the demands of most microbial species for this mineral nutrient.

The iron requirement of microorganisms may be intimately related to their ability to synthesize iron transport compounds, collectively designated siderochromes (Neilands, 1973). An interesting feature of the biosynthesis of both the hydroxamate- (Garibaldi, 1971) and the catechol- (Garibaldi, 1972) type siderochrome is the sensitivity of the process at higher temperatures. Thus a fluorescent pseudomonad grown above 28°C failed to form hydroxamate siderochromes and required addition of the latter to the medium for full cell yields. At 28°C the medium could be supplemented with either the siderochrome or high levels of inorganic iron, while at 20°C both additives had a marginal effect upon ultimate cells yields. Similarly, in Salmonella typhimurium the excretion of a catechol-type siderochrome, presumably enterobactin (enterochelin), is diminished as the temperature is raised from 31° to 36.9°C and at 40.3°C supplementation with iron transport compound is required. As pointed out by Garibaldi (1972), these observations may be relevant to the virulence of microbial species and to the value of fever as a defense mechanism. The role of iron in infection and virulence is considered in greater detail elsewhere in this volume (Bullen et al., this volume, Chapter 20); however, the work of Schneider (1967) and his colleagues is of historical interest and should be noted. He observed that the survival of mice which had been first injected with an avirulent strain of S. typhimurium and then challenged with a virulent strain of the same organism depended on dietary factors called pacifarins. Fermented, but not sterile, egg white proved to be a rich source of salmonellosis pacifarin. A strain of Aerobacter cloacae, as well as other enteric bacteria, were shown to form pacifarin when grown on egg white or on simple, synthetic media. A correlation was observed between the presence of iron chelating materials and the pacifarin potency of cultures; the addition of iron during growth of the organisms repressed pacifarin synthesis. Wawskiewicz et al. (1971) concluded that the cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine (enterobactin), but not the monomer, is active in the mouse salmonellosis model. The molecular explanation for the model is unresolved, but one obvious possibility is that the siderochrome stimulates growth of the avirulent strain which then immunizes the animal.

VI IRON LIGAND ATOMS IN MICROORGANISMS AND THEIR FUNCTION

Table I shows the range and diversity of iron-containing or iron-activated substances from microbial sources. This is certainly a minimal and provisional list; the full metabolic function of iron in any organism is not known. It will probably be first completely described in Escherichia coli and, indeed, a number of the iron-containing enzymes from this organism have been mapped on its chromosome (Fig. 3). This shows how much work remains to be done in order to understand the influx, anabolism, catabolism, and efflux (if any) of iron and its compounds in even the most thoroughly characterized microbe.

TABLE 1

Iron Proteins and Enzymes from Microorganisms

aIn the absence of a specific citation, reference to the component may be found in this volume. For reviews of the nonprotein iron compounds of microorganisms see Neilands (1966, 1972, 1973). Clostridial lysine 2, 3-aminomutase is activated by ferrous iron (Chirpich and Barker, 1971), yeast arginase is an iron metalloenzyme (Middlehoven, 1969), superoxide dismutase of the internal matrix of Escherichia coli is a manganoprotein while the enzyme in the periplasmic space is an iron protein (Gregory et al., 1973), and both assimila tory (E. coli) and dissimilatory (Desulfotomaculum nigrificans) sulfite reductases contain siroheme, an iron chelate of an eight carboxylate tetrahydroporphyrin (Murphy and Siegel, 1973).

bDonella et al. (1972) have proposed that phosvitinlike proteins shuttle iron from cytosol to mitochondria in the animal cell. For a discussion of the structure of iron phosvitin see Gray (1971).

Fig. 3 Relative location of some genes involved in iron metabolism in Escherichia coli. (1) Taylor (1970); Taylor and Trotter (1972); (2) Rosenberg and Young, this volume, Chapter 3; (3) Streicher and Valentine, this volume, Chapter 10; (4) Olle Karlström, personal communication; (5) Berberich (1972). Pop A, pop B and pop C, genes involved in porphyrin metabolism, are located at 11, 17 and 4 minutes, respectively (Taylor and Trotter, 1972). According to Pollack et al. (1970) an iron Operon, termed enb and apparently similar to the ent of E. coli, is located at about 20 min on the Salmonella chromosomal may (Sanderson, 1970). Twelve classes of Salmonella typhimurium mutants (sid), most of which map at 9 min, have been selected by use of a siderochrome antibiotic, albomycin (Luckey et al., 1972). The hem A, hem B, and pyr D genes have also been mapped in Salmonella.

Obviously, it is altogether naive, from a structural viewpoint, to classify iron derivatives according to the atom attached to the central metal ion. Such classification does not take into account stereochemical and other subtle conformational differences which may vastly influence the behavior of the complex in solution. It is attempted here only for the sake of convenience and to emphasize that all three small, commonly electronegative donor atoms, O, N, and S, are found in all possible bicombinations in microbial iron compounds.

The majority of iron compounds characterized from microbial sources belong to the iron transport family, or siderochromes (Neilands, 1973). The reasons for the relative wealth of information about this particular group of iron compounds may be summarized as follows: (a) The synthesis of the siderochromes is derepressed at low iron levels and it is a relatively straightforward exercise to make them, in the deferrated form, by simply withholding iron from the growth medium (Fig. 4). This finding (Garibaldi and Neilands, 1956), together with chemical, biological, and intuitive arguments, formed the basis for the proposal that the siderochromes function as iron carriers (Neilands, 1957). (b) The compounds have a molecular weight which is usually less than 1000 daltons, and a special technology has been worked out for their characterization (Emery, 1971). (c) The diversity of microbial DNA is responsible for the biosynthesis of variegated structures bearing phenolate, carboxylate, or hydroxamate groups in varying combinations, and (d) the structural latitude is wider for molecules which function in transport rather than in, for example, a multienzyme chain (Neilands, 1972).

Fig. 4 Typical repression of deferrisiderochrome production by iron observed in many microbial species growing on artificial media (Garibaldi and Neilands, 1956). Inset, total iron concentrations: (1) and (3), Bowen (1966); (2) and (4), Underwood (1971). The iron concentrations available for growth in these milieu may be many orders of magnitude below these figures; thus serum and milk contain well defined, very powerful ferric-binding proteins (Bullen et al., this volume, Chapter 20) and in such environments the genes programming the formation of the deferrisiderochrome-synthesizing enzymes will be substantially derepressed.

A All-Oxygen

The all-oxygen ligand is found commonly in the siderochromes, which is not surprising in view of the functional requirement in these compounds for a structure which will strongly and selectively bind ferric iron. The known and suspected siderochromes will not be listed here as the field has recently been reviewed (Neilands, 1973; Rodgers and Neilands, 1973). Suffice it to say that their biofunction has been proved through the rigorous and unambiguous methods of biochemical genetics (Pollack et al., 1970; Rosenberg and Young, this volume, Chapter 3). In addition, ferrichrome (Emery, this volume, Chapter 5), and mycobactin (Ratledge and Marshall, 1972). have been shown to have the capacity to transport iron in the source organisms for these siderochromes.

The siderochromes comprise all members of the ferrichrome series, which are cyclohexapeptides containing a tripeptide sequence of Nδ-acyl-Nδ-hydroxyornithine, as well as the ferrioxamines, the latter containing alternating units of succinic acid and 1-amino-ω-hydroxyaminoalkane. The siderochromes produced by enteric and other bacteria and which contain catechol groups coordinated to iron must also be placed in the all-oxygen ligand group. Not much is known about the coordination chemistry of members of the enterobactin (enterochelin) class, except that the iron coordination compounds are difficult to reduce. The latter contain the anion of catechol, which is easily oxidized to quinones which, in turn, polymerize into other products. Lillie (1972) quotes Link as using ferric gallate in what is possibly the first histochemical reaction, the demonstration of vascular canals in leaves (Lillie, 1972). Ferric gallate has long been employed as a coloring matter in ink and Tiron (disodium-1,2-dihydroxybenzene-3,5-disulfonate) is a useful reagent for ferric iron (Klotz, 1971; Phillips and Williams, 1966).

The ferric hydroxamates are extremely stable under a variety of conditions and certain members of the ferrichrome-type siderochromes have been investigated by X-ray diffraction, proton magnetic resonance spectroscopy, Mössbauer spectroscopy, and have been subjected to very high pressures. In spite of the large number of siderochromes isolated, numbering several dozen, ferrichrome A is the only one for which a crystal structure has been published although an X-ray diffraction structure of ferrichrysin has been completed (Rolf Norrestam, personal communication).

The existence of an X-ray structure for ferrichrome A suggested the possibility of an investigation of the solution conformation of the substance by means of high resolution PMR spectroscopy. The conformation of the main platform bearing the metal binding groups, the cyclohexapeptide moiety, can be ascertained by study of the six amide protons. Since the paramagnetic iron atom causes line broadening in the PMR signal, the ferric ion was replaced by diamagnetic ions such as aluminum and gallium. Although the ionic radius of aluminum (r0 = 0.53 Å) is further removed from that of gallium (r0 = 0.62 Å), than is the latter from ferric ion (r0 = 0.64 Å), the former two chelates gave identical PMR spectra. This indicates that the former are suitable models for the ferric complex. Four of the six peptide protons were found to have reduced slopes in the upfield shifts of their resonance positions with increasing temperature. By use of a series