JOURNAL OF CHEMOMETRICS

J. Chemometrics 2007; 21: 520528

Published online 4 September 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/cem.1058

Factor analysis of spectroelectrochemical reduction

of FAD reveals the pKa of the reduced state
and the reduction pathway
Edmund R. Malinowski1*, Michael J. Barber2, Graham T. Whitaker3 and Eugene T. Smith3
1

Stevens Institute of Technology, Hoboken, NJ, USA

University of South Florida, Tampa, FL, USA
3
Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, FL, USA
2

Received 10 January 2007; Revised 4 May 2007; Accepted 11 May 2007

The free flavin adenine dinucleotide (FAD) cofactor is known to exhibit a pH-dependent midpoint
potential involving a simultaneous two-electron transfer step (n 2). Uv-vis spectroelectrochemical
reductions of FAD at constant pH, ranging from 5 to 9, were recorded and analyzed by factor analysis.
Principal factor analysis was used to determine the number of species present at each pH. The results
indicate that only two composite forms of FAD are present: the oxidized and the reduced forms.
Window factor analysis was used to extract the concentration profiles of the controlling species. The
oxidized form was found to be a single pH-independent species, whereas the reduced form consists
of two species. The pH-dependent spectroscopic changes of reduced FAD were best modeled by a
single proton transfer step involving two different ionization states with an apparent pKa 6.3. This
value compares favorably with those obtained from NMR and from midpoint potential measurements. At pH 6, the reduction of FAD was found to be first order, whereas at pH 9 the reduction is zero
order; these observations are explained in terms of the reaction pathway involving xanthine oxidase,
its substrate, and the pH. Copyright # 2007 John Wiley & Sons, Ltd.
KEYWORDS: factor analysis; window factor analysis; Uv-vis spectroscopy; flavin adenine dinucleotide; FAD reduction

1. INTRODUCTION
Flavin adenine dinucleotide (FAD) is a well-characterized
coenzyme that is derived from riboflavin [1]. Flavoproteins
containing FAD carry out a broad range of biological redox
reactions, some involving the transfer of two electrons in
individual successive steps (i.e. two n 1 steps) or in a single
simultaneous step (i.e. an n 2 process). The FAD cofactor in
flavoenzymes is known to exist in three oxidation states (the
quinone, semiquinone, and hydroquinone), and each of these
oxidation states can exist in different ionization states. Over
the past half century, there have been numerous spectroscopic and electrochemical studies to identify different
ionization and oxidation states of free FAD, as well as
attempts to establish the reduction potentials and acid
dissociation constants associated with these species [16].
Notably, several spectroscopic studies of FAD [1,6], from pH
5 to 9, revealed no ionizable oxidized species and only a
single ionizable reduced species.

*Correspondence to: E. R. Malinowski, 5042 SE Devenwood Way,

Stuart, FL 34997, USA.
E-mail: malinowski@comcast.net

In one of the earlier electrochemical studies [2], Lowe and

Clark analyzed the reduction potential as a function of pH in
an attempt to establish acid dissociation constants and
reduction potentials of the free FAD cofactor. Equilibrium
constants for proton and electron transfer steps of various
flavoproteins have also been reported [7] using the
methodology popularized by Clark [8]. A number of
simplifying assumptions were made in this methodology,
and the acid dissociation constants were derived from
inflection points in reduction potential versus pH curves. In
contrast, Smith and coworkers [912] developed a model that
explicitly treats each electron and proton step, and have
demonstrated that there are numerous equilibrium constants
that reproduce the experimental reduction potential versus
pH data. These studies clearly show that it is not possible to
assign values to these equilibrium constants without
additional information concerning the oxidation and ionization states of FAD as a function of solution potential and pH.
In this study, we present spectroscopic data as a function
of pH. In order to identify the different ionization and
oxidation states of FAD the experimental information was
subjected to a number of statistical methods collectively
known as factor analysis. This methodology is routinely used
Copyright # 2007 John Wiley & Sons, Ltd.

Factor analysis of FAD reduction reveals pKa of reduced state and reduction pathway 521

to determine the probable number of species responsible for

the spectroscopic data, and to extract the spectra of the
underlying species as well as their concentration profiles
[13]. Despite its use in other chemical disciplines, factor
analysis has not been used extensively for the analysis of
spectro-electrochemical data [14]. Factor analysis is not based
on any chemical model, rather it uses mathematical and
statistical methods to reduce large data sets to their lowest
dimensionality and to explore the underlying nature of the
intrinsic factors without relying on preconceived models of
the system. In this study, principal factor analysis (PFA) is used
to determine the number of spectroscopically distinguishable species. A special technique called window factor analysis
(WFA) is used to establish concentration profiles, providing
information that offers valuable clues concerning the
identities of the underlying species (i.e. oxidation and
ionization states) and the controlling equilibria. From such
information thermodynamic parameters are deduced as well
as the number of protons and electrons involved in the
electron transfer process.

deviation in the measurements is closest to the RSD obtained

from the factor analysis.
After determining the number of primary factors, the
concentration of each individual component may be determined via WFA. Window factor analysis is a model-free
method designed to extract the concentration profiles of
individual components from evolutionary processes originating from titrations or kinetic experiments [13]. This
technique takes advantage of the fact that each component
lies in a specific region along an evolutionary axis, referred to
as a window. The method, however, requires that all other
components have signals outside the window although they
may also have signals inside the window. In this application,
window factor analysis was applied to the visible spectra
recorded as a function of pH. The concentration profiles of
the different ionization states of the chemical species as a
function of pH were determined using this method.

3. EXPERIMENTAL
3.1. Sample preparation

2. THEORY
Factor analysis encompasses a number of mathematical and
statistical methods. It is used to analyze large data sets
without relying upon any preconceived chemical model. In
many applications, PFA is first used to determine the number
of controlling factors involved in a set of data. Details of the
chemical system must be known in order to exclude
experimental artifacts that may appear as unique factors.
There are a number of complicating issues such as detector
noise, baseline drift, signal nonlinearity, data pretreatment
effects, sampling error, etc. Such undesirable effects often
confuse the analysis because they produce ghost factors that
have no chemical meaning. Noise is a component of all
experimental data sets, and therefore care must be taken to
exclude this artifact as a chemical species. Also, it is
important to note that the number of chemical species
present is not necessarily the same as the number of factors.
For example, if the concentrations of two or more species
change but remain in the same ratio due to chemical
equilibrium only a single factor will be observed for these
multiple components.
A variety of factor analytical techniques have been
developed to determine the number of significant factors
responsible for a data matrix, methods such as the residual
standard deviation, the factor indicator function, F-tests for
establishing significance levels, distribution of misfit,
chi-squared tests, cross validation, etc. [13] The residual
standard deviation (RSD), also known as the real error (RE) is
calculated from the eigenvalues extracted from the data
matrix. The primary factors are associated with the largest
eigenvalues, accounting for the maximum variation in the
data. The secondary factors are associated with the smallest
set of eigenvalues, accounting for the noise as well as other
undesirable instrumental and experimental artifacts. If a
reasonable estimate of the noise in the data (i.e. the standard
deviation) can be determined prior to the factor analysis the
number of primary factors can be determined. The number of
factors corresponds to the point where the standard
Copyright # 2007 John Wiley & Sons, Ltd.

Flavin adenine dinucleotide disodium salt hydrate (Sigma

Aldrich, St. Louis, MO) was suspended in a 50 mM buffer:
acetate, pH 55.5; MES (2-(4-Morpholino)ethanesulfonic
acid), pH 66.5; phosphate, pH 6.57, TRIS, pH 7.58; and
borate, pH 9. Because the experiments were conducted over
the course of a year with different FAD stock solutions, the
initial concentrations of FAD in each buffer could not
be controlled precisely and varied by as much as 10%.
This experimental artifact introduced an especially challenging problem for chemometric analysis. The resulting
solution, which was approximately 120 mM in FAD
(e 11.3 cm
1mM
1 at 450 nm), was degassed and placed
in a Coy anerobic chamber maintained at <1 ppm oxygen.
The FAD solution was reduced by the xanthine/xanthine
oxidase redox reaction according to a modified method
developed by Massey [15]. Xanthine was prepared by
dissolving 15 mg of xanthine in 100 mL of 1 M NaOH and
then diluting to 10 mL with deionized water. This mixture
was added to the FAD solution to yield a final concentration
of 900 mM xanthine. To initiate reduction, an appropriate
amount of xanthine oxidase was added to the mixture to
bring about complete reduction in 1220 h (the amount of
enzyme added was pH-dependent, but was typically around
50 nM). No redox dyes were used since they would overlap
with the spectrum of FAD.

3.2. Spectroscopic measurement

Uv-visible spectra were collected every 5 min for 20 h using
an Ocean Optics spectrometer fitted with a deuterium lamp
and thermostated CCD detector (S2000 TR) set at 258C.
Reference and dark spectrum were collected prior to each
trial. Because of the long time required to complete any single
experiment the reference spectrum and the dark spectrum
changed somewhat with time despite thermostating the
detector. These uncontrollable variables produced additional
uncertainties in the absorption measurements. One hundred
spectra were averaged over 8 milliseconds, and the boxcar
average was set at 2. Emission lines of the source caused
aberrations in the spectra at 487 and 437 nm. The measured
J. Chemometrics 2007; 21: 520528
DOI: 10.1002/cem

522 E. R. Malinowski et al.

Table I. Residual standard deviations, RSD(n), obtained from PFA (showing only the first three residuals) compared to the
standard deviations, STD, estimated from the isosbestics point at 340 nm for the buffered solutions FADpHx, and from the baseline
at 550 nm for FADox and FADred matrices. The number of chemical factors, n, corresponds to the RSD level that is closest to the
estimated STD
Data Matrix

STD (10
3)

RSD(1) (10
3)

RSD(2) (10
3)

RSD(3) (10
3)

FADpH 5.0
FADpH 5.5
FADpH 6.0
FADpH 6.2
FADpH 6.5
FADpH 6.7
FADpH 7.0
FADpH 7.5
FADpH 8.0
FADpH 9.0
FADox
FADred

8.56
4.22
4.35
4.75
2.65
1.60
3.47
3.00
1.94
2.22
18.79
15.73

93.41
113.48
93.68
116.45
128.50
125.29
135.97
140.88
116.71
108.35
15.47
31.12

3.06
3.54
2.63
2.56
2.12
2.62
2.58
2.48
2.15
2.34
10.12
12.71

1.97
0.69
0.85
1.15
0.88
0.92
0.74
0.75
0.88
0.62
8.05
5.55

2
2
2
2
2
2
2
2
2
2
1
2

absorbances were not corrected for baseline nor for variations in the initial concentrations of FAD.

3.3. Data sets

Twelve data matrices were compiled from 10 experiments
conducted at 10 different pHs listed in Table I. The first 10
matrices contain the spectral absorbances, from 320 to
550 nm, recorded during the kinetic experiment, each
conducted at a constant pH. For convenience these matrices
are labeled FADpHx, where x is the pH value. Throughout
this paper, bold letters will be used to indicate matrices and
vectors. Two special matrices were compiled from this pool
of data. The very first spectrum recorded at each pH was
assembled into a matrix to represent the oxidized form of
FAD as a function of pH. The very last spectrum at each pH,
where it appeared that the reaction was complete (no further
change in absorbance), was assembled into a matrix to
represent the reduced form of FAD as a function of pH. These
two matrices are labeled FADox and FADred.

factors. This is to be expected, because these techniques are

sensitive to baseline drift, fluctuations in the light source,
emission from the deuterium source, temperature variations,
etc.
The compiled matrices, FADox and FADred, as described
in the Experimental Procedures Section, are shown in
Figure 2. Neither data sets exhibit discernable isosbetics
points due to relatively large experimental uncertainties. In
order to determine the number of chemical factors responsible for these two data sets, the standard deviations in the
baselines at 550 nm were used to estimate the uncertainty in
the absorbance measurements. Because these matrices
involve spectra generated from 10 differently buffered pH
solutions the uncertainties associated with these matrices are
expected to be much larger than those associated with their
parent solutions. The marked increases in the standard
deviations (STD) are displayed in Table I. Comparing STD to
the RSD(n) obtained from PFA we conclude that only one

4. RESULTS
4.1. Principal factor analysisnumber
of factors
The reduction of FAD at pH 7 as a function of time is
illustrated in the spectra of Figure 1. Similar data were
obtained at other pHs. The noise in the data was quantified
by determining the standard deviation (STD) about the
isosbestic point located at 340 nm. Values obtained as a
function of pH are provided in Table I. The absorption data
were then analyzed by PFA yielding RSD(n), at each pH as a
function of factor level, n. Table I displays only the first three
residuals, all other residuals are superfluous because they
systematically decrease as the factor level increases. At each
pH, the standard deviation estimated from the isosbestic
point is closest to RSD(2) corresponding to the two-factor
level, giving evidence that two factors account for each
FADpH data matrix. It is interesting to note that all other
common criterion [13] for determining the number of
relevant factors, such as F-tests, cross validation, the factor
indicator function, etc., yielded an excessive number of
Copyright # 2007 John Wiley & Sons, Ltd.

Figure 1. Reduction of 122 mM free FAD in 200 mM phosphate at pH 7. Individual spectra were collected every 5 min
intervals but only selected spectra are shown. The isosbestics
point at 340 nm was used to determine the standard deviation
in the absorbance measurements.
J. Chemometrics 2007; 21: 520528
DOI: 10.1002/cem

Factor analysis of FAD reduction reveals pKa of reduced state and reduction pathway 523

Figure 2. (A) FADox: Visible spectra of oxidized FAD from

pH 5 to pH 9 (approximately 120 mM in FAD) (first spectrum of
each experiment). (B) FADred: Visible spectra of reduced
FAD from pH 5 to pH 9 (last spectrum of each experiment).

Figure 3. Concentration profiles extracted by window

factor analysis. (A) At pH 6 the profiles conform to a first-order
reaction. (B) At pH 9 the profiles conform to a zero-order
reaction.

factor is responsible for FADox, whereas two factors are

responsible for FADred.
The signal-to-noise ratio (S/N) of the absorbance measurements was estimated by dividing the root-mean-square of
the absorbances by the STD. For all 10 data matrices this
yielded S/N 200.

coefficient of the semiquinone is of the same order of

magnitude as the oxidized and reduced forms of FAD, as
shown by Massey [16]. Consequently, interpretation of the
data is limited solely to the behavior of the oxidized and
reduced species. As illustrated in Figure 3, the concentration
profiles of FAD are first order at pH 6 and zero order at pH 9.
This unexpected anomaly gives evidence for the reduction
pathway presented in the Discussion Section.
WFA cannot be applied to FADox because these data
originate from a single component, independent of pH. All
attempts to apply WFA to an expanded factor space (n 2, 3,
etc.) produced indecipherable scatter plots. The differences
in the spectra shown in Figure 2A result from differences in
both the initial concentrations of the FAD and the chemical
nature of the buffers.
Based on two principal factors, the reduced spectra of
FAD, FADred, were analyzed by window factor analysis,
which yielded the concentration profiles shown in
Figure 4A. This figure provides us with valuable clues that
help us decipher the equilibrium between the reduced
species, as explained in the Discussion Section.

4.2. Window factor analysisconcentration

profiles
The absorption spectra of the 10 FADpHx experiments,
described in the Experimental Procedures Section, were
subjected to window factor analysis using two factors as
determined by PFA. We found no evidence for the existence
of any measurable amount of semiquinone. Although the
semiquinone may play an important role in the reduction
process, its presence is below the detection level of the
spectroscopic measurements of this investigation. The
noise-to-signal ratio is the reciprocal of S/N. Because
S/N 200 the semiquinone concentration is estimated to
be less than 0.5%, assuming, of course, that the extinction
Copyright # 2007 John Wiley & Sons, Ltd.

J. Chemometrics 2007; 21: 520528

DOI: 10.1002/cem

524 E. R. Malinowski et al.

that two factors account for each absorbance matrix. These

two factors are associated with the oxidized and the reduced
forms of FAD. The appearance and subsequent disappearance of semiquinone would yield three principal factors
instead of two. Our analysis indicates that no measurable
amount of semiquinone was produced during the
reductions. Principal factor analysis was also carried out
on the fully oxidized spectra (FADox) and the fully reduced
spectra (FADred). These matrices are functions of pH. PFA
analysis indicates that the oxidized spectra involve a single
factor, whereas the reduced spectra involve two factors. The
reduced spectra were then subjected to WFA. The concentration profiles of the two underlying components of FADred
were extracted by WFA. It is important to note that the
extracted concentrations are not based on any chemical
model, they are simply the result of the mathematical
analysis which takes advantage of the evolutionary nature of
the reductions.

5.2. Chemical modeling

Figure 4. Results obtained from window factor analysis of

the reduced FAD data matrix, FADred. (A) Concentration
profiles of the underlying components. (B) Spectra of two
underlying components.

The spectra of the two underlying components of reduced

FAD were obtained by applying Beers law:
A SC

(1)

where A is the absorbance matrix, S is the component spectra

and C is the component concentrations. This expression can
be rearranged to
S AC

(2)

where CR is the pseudoinverse of C. According to

Equation 2 postmultiplying FADred by the pseudoinverse
of the concentration profile matrix displayed in Figure 4A
yielded the spectra of the components displayed in
Figure 4B.

5. DISCUSSION
5.1. Factor analysis
The FADpHx data matrices, described in the Experimental
Procedures Section, conducted at 10 different pH, were
subjected to principal factor analysis (PFA). PFA revealed
Copyright # 2007 John Wiley & Sons, Ltd.

Recall that only two factors account for each of the 10

FADpHx matrices and that no spectroscopic evidence for the
semiquinone was observed. Because the oxidized species are
in equilibrium throughout each experiment, conducted in a
buffered pH system, their concentrations will decrease in the
same proportion producing a composite spectrum that does
not vary even though several different oxidant species may
be present. Similarly, the reduced species produces a
composite spectrum that does not vary throughout the
experiment. Thus, only two spectroscopically distinguishable composite species are observed for each of the FADpHx
matrices.
The above analysis dealt with spectral changes as a
function of solution potential at a specified pH. Spectral
changes as a function of pH were also considered by
assembling the initial spectra of the reductions into matrices
FADox and FADred. Because these matrices involve
different pHs their spectroscopic composites depend upon
the pH as dictated by the dissociation equilibria. In the
present study only one factor was determined for FADox;
this is consistent with previous studies that report that the
neutral form is the only species present below pH 10 [1].
PFA indicates that two factors are responsible for FADred.
The concentration profiles of two chemical species extracted
by WFA are shown in Figure 4A. The crossover point at pH
6.3 corresponds to the situation where the concentrations of
the two species are equal. The experimental data were then
compared to theoretical models involving multiple protons
since FAD is known to exist in different ionization states.
The chemical reaction and equilibrium constant for a
single proton transfer step are represented as follows:
RH R H and Ka RH =RH
When [R] [RH], Ka [H], hence pKa pH. Thus the
crossover point at pH 6.3 in Figure 4A implies that pKa 6.3.
In comparison, the associated reaction and equilibrium
constant for a two-proton transfer step are given by
RH2 R 2H and Ka RH 2 =RH2 
J. Chemometrics 2007; 21: 520528
DOI: 10.1002/cem

Factor analysis of FAD reduction reveals pKa of reduced state and reduction pathway 525
2

When [R] [RH2], Ka [H ] , hence pKa 2 pH. In this case,

Figure 4A implies that pKa 12.6.
These equilibrium expressions were used to calculate
theoretical concentrations as a function of pH, illustrated by
smooth lines in Figures 5. The jagged black lines in the figure
connect the model-free concentrations obtained from WFA
(Figure 4A). The point-by-point sum of squares of the
differences between the theoretical concentrations and WFA
concentrations were found to be 0.20 for the single-proton
transfer model and 0.50 for the two-proton transfer model.
Thus, the model involving a single proton transfer with a pKa
equal to 6.3 is more consistent with the experimental data.

5.3. Reconstruction of spectra

Recall that the spectra of the raw data are displayed in
Figure 2. Absorbances of FAD species can be predicted from
the theoretical concentration profiles by means of Beers law
as shown in Equation (2). Premultiplying the theoretical
component concentrations, C, displayed in Figure 5 by the
pure component spectra, S, displayed in Figure 4B, gives the
smooth curves in Figure 6A.
The spectra of the raw data displayed in Figure 2B appear
to be substantially different than those based on the
theoretical model shown in Figure 6A. This difference is
caused by two important experimental artifacts: (1) variations in the initial concentrations of FAD and (2) baseline
shifts due the different buffered solutions. These artifacts can
now be accounted for by finding a suitable set of coefficients,
mj and bj, to convert the raw spectra, A(raw), into those
obtained from window factor analysis, A(wfa), in accord
with the following expression:
Aij wfa mj Aij rawbj

(3)

where Aij is the absorbance at wavelength i and pH j. Here mj

corrects the raw spectra for differences in the initial
concentrations and bj corrects for differences in the baselines.
These two coefficients are treated as functions of pH only.

Figure 6. Predicted spectra of reduced FAD based on (A)

Beers law, A SC, (Equation (1)), and (B) concentration and
baseline corrected data according to Aij(wfa) mjAij(raw) bj
(Equation (3)).

Applying a method of least squares to the reduced data

matrix FADred we obtained the coefficients displayed in
Table II. Using these coefficients, the raw spectra were then
converted into the corrected spectra shown in Figure 6B.

Table II. Coefficients obtained from least-squares analysis of

FADred and FADox using Aij (wfa) mjAij (raw) bj
(Equation (3)) and Aij (ave) mjAij (raw) bj (Equation (4))

Figure 5. A comparison of the window factor analysis results

(ragged lines) and the theoretical profiles (smooth curves)
based on (A) a single proton transfer with pK 6.3 and (B) a
simultaneous two-proton transfer with pK 12.6.
Copyright # 2007 John Wiley & Sons, Ltd.

pH

FADred mj

FADred bj

FADox mj

FADox bj

5.0
5.5
6.0
6.2
6.5
6.7
7.0
7.5
8.0
9.0

0.968
1.041
1.037
1.001
0.991
0.928
0.949
0.929
1.038
1.055

0.0208
0.0327
0.0100

0.0142

0.0060

0.0138
0.0135
0.0101

0.0229
0.0181

1.016
1.035
1.036
1.008
0.981
0.909
0.990
0.921
1.039
1.089

0.0347
0.0226

0.0066

0.0152

0.0214
0.0023
0.0189
0.0203

0.0164
0.0339

J. Chemometrics 2007; 21: 520528

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526 E. R. Malinowski et al.

Notice that the corrected spectra exhibit clearly defined

isosbestics points, as expected. It is interesting to note that
when the spectra displayed in Figure 6B was subjected to
WFA the sum-of-squares of the differences between the
theoretical and calculated concentrations decreased from
0.20 to 0.10, representing a significant improvement in the fit,
as expected.
The FADox spectra illustrated in Figure 2A can also be
converted into its average spectra, A(ave) using a similar
expression, namely

constant equal to 10
6.3), two possible reduction pathways,

labeled Path 1 and Path 2, as depicted by Equations. (5) and
(6), are possible.
Path 1:
FAD 2e
FAD2

(5a)

FAD2
H FADH

(5b)

FAD H 2e
FADH

(6a)

Path 2:

Aij ave mj Aij rawbj

FADH H FADH2

(4)

where Aij(raw) is an element of FADox. Equation (4)

involves the average spectra because the spectra of the
oxidized species are independent of pH. (Recall that PFA
gives evidence for only one oxidized species.) The coefficients obtained from this procedure are listed in Table II. This
set of coefficients converts the oxidized data matrix into the
spectra shown in Figure 7. The values of mj from both
procedures are almost identical, following the same
variation. This fact supports our contentions that the
differences between Figure 2 and the reconstructed spectra
are primarily due to differences in the concentrations of FAD.
Furthermore, from Table II we see that the initial concentrations of the solutions differ by as much as 18%. Little or no
correspondence is expected between the bj coefficients
because these values incorporate baseline drift as well as
baseline shift due to solvent differences. However, it is
interesting to note that the mean-square values of the bj
coefficients, 0.0216 for FADox and 0.0178 FADred, compare
very favorably with their standard deviations, 0.01879 and
0.01573, as listed in Table I.

5.4. Reduction potentials

The absolute number of protons associated with oxidized
and reduced forms of FAD in many flavoproteins are
unknown [17]. Using the information gleaned from factor
analysis (i.e. a single oxidized species, no observable
semiquinone and two reduced species with a dissociation

(6b)

The first steps in Paths 1 and 2, (5a) and (6a), represent the
ionization which involves reduction of FAD by xanthine
(Xan) catalyzed by xanthine oxidase (XanOx(cat)). The
second steps, (5b) and (6b), represent single proton transfers.
Overall, Path 1 involves a single proton transfer, whereas
Path 2 involves two proton transfers, the reduction step
accounting for one of the transfers. Both paths are
hypothetically possible. Both mechanisms assign the dissociation constant, 10
6.3, to the second step, which differs
stoichiometrically for each model. We will, however, present
evidence to show that Path 2 represents the correct
mechanism.
These models yield two different expressions for the
half-cell reactions as shown in Equations (7) and (8).
According to Nernst theory the midpoint reduction potential, Emid, is a function of the standard reduction potential, Eo,
the dissociation constant, Ka, and the hydrogen ion
concentration as shown in Equations (7) and (8).
Path 1:
Eo 59
Ka

log
2
2
Ka H 

(7)

Eo 59
Ka

log
2
2
Ka H  H 2

(8)

Emid
Path 2:
Emid

The paper of Lowe and Clark [2] provides us with

information that can be used to test these models.
Experimental values of the midpoint reduction potentials
of FAD as a function of pH, taken from reference [2], were
inserted into Equations. (7) and (8), together with Ka 10
6.3,
yielding standard reduction potentials. The average values
for Eo were determined to be
453 mV for Path 1 and
30 mV
for Path 2. The midpoint potentials were then recalculated
by inserting the respective average values of Eo into
Equations. (7) and (8). The results are displayed in
Figure 8, together with the experimental values. This plot
gives clear evidence that Path 2 represents the correct
mechanism. This agrees with the observations of Lowe and
Clark [2] who concluded that the free cofactor exhibits a
pH-dependent reduction potential accompanied by a
two-electron transfer (n 2) in a single step involving the
addition of a proton.

5.5. Reduction pathway

Figure 7. Predicted spectra of oxidized FAD based on Aij(ave) mijAij (raw) bj (Equation (4)).
Copyright # 2007 John Wiley & Sons, Ltd.

The switching from first-order to zero-order reduction as the

pH increases from 5 to 9 can be explained in terms of the
J. Chemometrics 2007; 21: 520528
DOI: 10.1002/cem

Factor analysis of FAD reduction reveals pKa of reduced state and reduction pathway 527

Equation (11) yields

1 FAD0 b
FAD0
FAD t
ln
a FAD a

(12a)

a kKOH
cato

(12b)

b KOH


(12c)

where

and

Figure 8. Midpoint reduction potentials of FAD as a function

of pH. The squares represent experimental values taken from
reference 2. The size of the squares emulate the experimental
errors,  0.1 pH and  5 mV. The steep curve was determined
using theoretical Path 2 according to Equation (8). The nearly
horizontal curve results from Equation (7), Path 1.

reagents involved. The reduction of FAD is accompanied by

the oxidation of xanthine, as shown in Equation (9)

(9)
The hydrogen ion and the two electrons released from this
half-cell are used to reduce FAD (see Equation (6a)). The
overall reaction is initiated by xanthine oxidase catalyst,
XanOx(cat), which produces a steady-state intermediate
leading to the final products. The oxidation-reduction
process can be depicted as
FAD Xanthine OH
XanOxcat
Intermediate ! FADH
UricAcid

(10)

The formation of the intermediate is reversible but its

decomposition into products is irreversible. The exact nature
of the intermediate cannot be determined from the spectroscopic data obtained in this investigation. Application of
steady-state theory leads to the following expression for the
rate of reduction of FAD:
dFAD
kKOH
FADcatO


1 KOH
FAD
dt

(11)

In this equation k is the limiting rate constant controlling

the formation of the reductants via catalytic intermediates,
K Xano =KM where KM is the Michaelis constant and
[Xan]o is the total concentration of xanthine, which does
not vary because it is in excess, and cato is the total
catalytic concentration of xanthine oxidase. Integration of
Copyright # 2007 John Wiley & Sons, Ltd.

The first term on the left of Equation (12a) represents a

first-order contribution; the second term represents a
zero-order contribution. Thus, the single pathway predicts
a mixed-order reaction. It is interesting to note that at high
pH this expression reduces to a zero-order reaction, and at
low pH it reduces to first order. A series of simulation studies
based on Equation (12) were carried out using the above
information together with the experimental concentrations of
the reagents. By setting K 1012 we were able to produce
concentration profiles similar to those shown in Figure 3 as
well as the profiles obtained from WFA at all other pHs. The
profile shapes are independent of the rate constant k because
this term appears in the denominator of both terms in
Equation (12a).

6. CONCLUSION
This investigation shows that factor analysis can yield
valuable information regarding the reduction of FAD. The
advantage of this method is that it can resolve a complex
system without imposing any prior assumptions about the
system. As clearly illustrated here, the method can be used to
extract information from experiments that are difficult to
control all of the variables, especially during sample
preparation.
The results obtained are consistent with those obtained by
investigators using quite different techniques. For example,
from 15-N chemical shifts Franken and coworkers [18] found
that the pKa of the reduced form of pure flavin mononucleotide (FMN) has a value of 6.8. This is in good agreement with
the pKa value of the reduced form of pure FAD determined to
be 6.3 in the present investigation. The difference in pKa
values may be due to the chemical difference between FMN
and FAD, or due to some artifact of the different techniques
used to obtain these values. Exact agreement is not to be
expected between the pKa determined in this study for FAD
and the pKa previously determined for FMN. At the present
time there is no known way to account for the effects of
different kinds of buffers on the spectra of the FAD species.
Their effects appear to be very small and have been ignored
in the present investigation as well those of Lowe and Clark
[2], Mayhew [5], and many others.
The proposed kinetic mechanism is also consistent with
the spectroscopic observations. It explains the switching
from first-order to zero-order reaction rate as a function of
pH. The nature of the intermediate cannot be determined
from these measurements because its signal is below the
detection limits of the instrument. It is tempting, however, to
speculate that the elusive semiquinone plays an important
role in the process.
J. Chemometrics 2007; 21: 520528
DOI: 10.1002/cem

528 E. R. Malinowski et al.

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J. Chemometrics 2007; 21: 520528

DOI: 10.1002/cem