REVIEW ARTICLE
Deflavination and reconstitution of flavoproteins
Tackling fold and function
Marco H. Hefti*, Jacques Vervoort and Willem J. H. van Berkel
Laboratory of Biochemistry, Wageningen University, the Netherlands
Flavoproteins are ubiquitous redox proteins that are
involved in many biological processes. In the majority of
flavoproteins, the flavin cofactor is tightly but noncovalently
bound. Reversible dissociation of flavoproteins into apo-
protein and flavin prosthetic group yields valuable insights in
flavoprotein folding, function and mechanism. Replacement
of the natural cofactor with artificial flavins has proved to be
especially useful for the determination of the solvent acces-
sibility, polarity, reaction stereochemistry and dynamic
behaviour of flavoprotein active sites. In this review we
summarize the advances made in the field of flavoprotein
deflavination and reconstitution. Several sophisticated
chromatographic procedures to either deflavinate or
reconstitute the flavoprotein on a large scale are discussed. In
a subset of flavoproteins, the flavin cofactor is covalently
attached to the polypeptide chain. Studies from riboflavin-
deficient expression systems and site-directed mutagenesis
suggest that the flavinylation reaction is a post-translational,
rather than a cotranslational, process. These genetic
approaches have also provided insight into the mechanism
of covalent flavinylation and the rationale for this atypical
protein modification.
Keywords: apoprotein; deflavination; FAD; flavin; flavo-
enzyme;flavoprotein;FMN;(metal)affinitychromato-
graphy; reconstitution.
Introduction

dehydrogenase [34].
In this paper, we present an overview of the field of
flavoprotein deflavination and reconstitution. This topic is
of central interest to flavin enzymology as it provides
valuable insights in flavoprotein folding, function and mech-
anism. Earlier reviews in this field have concentrated on the
methods of apoflavoprotein preparation [35–37], the use of
artificial flavins [38], and the functional role and mechanism
of covalent flavinylation [8,39]. Here, new methods of
flavoprotein reconstitution are described and combined
with insights obtained from the structural and functional
analysis of mutant enzymes. In the first part of this review,
the thermodynamics of flavin binding and the value of flavin
analogs are briefly discussed. Then, the old vs. new
approaches of reversible flavin removal are evaluated.
Finally, attention is given to the reconstitution of proteins
containing covalently bound flavins. A comprehensive
overview of the kinetics and thermodynamics of flavo-
protein reconstitution is beyond the scope of this article
and only selected cases are discussed.
Correspondence to W. J. H. van Berkel, Laboratory of Biochemistry,
Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
the Netherlands. Fax: + 31 317 484801,
E-mail:
Abbreviations: DAO,
D
-amino-acid oxidase; HAP, hydroxyapatite
chromatography; VAO, vanillyl-alcohol oxidase; PHBH, p-hydroxy-
benzoate hydroxylase; Nbs2, 5,5¢-dithio-bis(2-nitrobenzoate);
IMAC, immobilized metal-affinity chromatography.

the contribution of the phosphate to the binding energy is
the greatest (7 kcalÆmol
)1
), that the contribution of the
isoalloxazine is around 5–6 kcalÆmol
)1
, and that the ribityl
side chain contributes only 1 kcalÆmol
)1
. For flavodoxin
from Desulfovibrio vulgaris it was found that riboflavin only
binds to the apoprotein in the presence of inorganic
phosphate (Fig. 1). Moreover, co-operative effects were
observed linked to the binding of inorganic phosphate and
the 5¢-phosphate of FMN [48]. It was proposed that
phosphate binding induces a conformational switch,
creating a population of apoflavodoxin that is capable of
binding the isoalloxazine ring [49].
The thermodynamics of FAD binding to
D
-amino-acid
oxidase (DAO) has been studied by Matteo and Sturtevant
[40]. The free energy of binding was shown to be largely
independent of temperature. However, the enthalpy and the
entropy of the binding interaction were strongly tempera-
ture dependent. In contrast to the binding of FMN to
apoflavodoxin, where the entropy strongly opposes bind-
ing, the binding of FAD to DAO is enforced by a large
positive entropic contribution. It was proposed that this is
due to a decrease in the exposure of nonpolar groups to the

contribution in the preparation of apoflavoproteins. Many
flavoproteins are less stable when they loose their cofactor
and care should be taken when removing the flavin
prosthetic group. An attractive deflavination approach is
to bind the protein to a chromatographic support. This
diminishes entropic contributions by increasing the rigidity
of the apoprotein.
Flavin analogs
In order to gain insight into how the protein environment
influences the reactivity of the flavin, it is desirable to
remove the native prosthetic group in a, for the protein,
nondestructive way. The flavin prosthetic group can be
replaced with an artificial [38,52–56] or isotopically enriched
analog [57–69]. Replacement with a flavin analog should
result in the (functionally active) reconstituted holoprotein.
FMN and FAD analogs can be synthesized conveniently
from riboflavin, either chemically [1] or enzymatically [3],
and can be isotopically enriched [70].
Fig. 1. Crystal structure of Desulfovibrio vulgaris flavodoxin. The
protein is depicted in green, the riboflavin moiety of FMN in yellow
and the 5¢-phosphate moiety in lime green.
4228 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Artificial flavins have proved very useful in the determin-
ation of the solvent-accessibility, polarity, reaction stereo-
chemistry, and dynamic behaviour of flavoprotein active
sites [38]. Artificial flavins can be used to study the relative
distance between particular flavin atoms and the protein,
especially when using a series of flavins which are modified
at a specific position, thus varying the Vanderwaals radius
of the substituent. The solvent accessibility of the dimethyl-

Flavin analogs modified in the ribityl side chain may also
provide insight into flavoprotein function and mechanism
[54,84–87]. For medium-chain acyl-CoA dehydrogenase,
it was shown that the replacement of natural FAD by
2¢-deoxy-FAD reduces the activity of the enzyme about a
millionfold [84]. This strongly supported the view that the
2¢-hydroxyl group of the flavin ribityl chain is involved in
the stabilization of the partial negative charge of the
carbonyl oxygen of the acyl-CoA substrate in the transition
state [84,88,89].
Isotopically enriched flavins are suitable to get a detailed
view into the molecular and submolecular structure of the
protein-bound flavin molecule.
13
Cand
15
NNMRchemical
shifts can reveal both p electron density, conformational
changes and dynamic behaviour of the flavin moiety, as well
as the presence of specific hydrogens at the carbon and
nitrogen atoms investigated.
13
Cand
15
N have a natural
abundance of 1.1% and 0.4%, respectively. Therefore, the
flavoprotein has to be reconstituted with
13
C- and
15

for flavin removal should be relatively short.
As each (apo)flavoprotein has its own characteristics,
several strategies for the reversible removal of flavins from
flavoproteins have emerged. Initial deflavination protocols
were based upon precipitation, partial unfolding, or dialysis
of the protein [35]. More recent techniques focus on the
binding of the protein to a chromatographic support,
facilitating the removal of the flavin, and reconstitution of
the apoprotein [36,92].
When studying the properties of apo or reconstituted
flavoprotein, one needs to consider the side-effects of
residual flavin in the endproduct. If replacement with a
flavin analog is desired, residual natural flavin might
influence the catalytic properties of the reconstituted enzyme
considerably. The presence of residual flavin can even be
more problematic when investigating the physical and
spectroscopic properties of the apoprotein.
Below we describe several apoflavoprotein preparation
procedures, starting with conventional methods. Then we
turn to the growing field of immobilization-based deflavin-
ation methods in which one uses specific characteristics of
the holo flavoprotein to obtain the corresponding apopro-
tein. As a guide, the methods of apoflavoprotein prepar-
ation are listed in Table 1.
Conventional methods
In 1935, Theorell was the first who reported that flavopro-
teins could be reversibly resolved into their constituents
apoprotein and prosthetic group. To weaken the binding of
the flavin, Old Yellow Enzyme was dialysed at pH 2.7, thus
releasing the noncovalently bound FMN [93]. A few years

the related FMN-binding domain of cytochrome P450 BM3
[103]. The precipitated apo forms of both these proteins can
withstand the extreme acidic conditions applied and dissolve
readily at neutral pH.
Many flavoproteins irreversibly aggregate at low pH.
Therefore, a procedure of apoprotein preparation was
developed based on dialysis against halide anions at physio-
logical pH. Flavoproteins which bind the flavin prosthetic
group rather weakly can be deflavinated using a high
concentration of bromide ions [104–110]. Chloride is less
chaotropic and therefore less effective in removal of the flavin
[111]. Stronger chaotropes such as cyanide, cyanate and
thiocyanate have been used as well, but with these nucleo-
philic agents significant conformational perturbations pre-
venting holoprotein reconstitution may occur [109,112,113].
Addition of a phosphodiesterase or phosphatase to dilute
solutions of holoflavoprotein shifts the equilibrium to the
apo form, because free FAD or FMN is hydrolysed to
FMN and riboflavin, respectively. These reactions are
relatively fast, but not very useful for large scale apoprotein
preparation [114]. Moreover, care must be taken to remove
the cleavage enzyme.
Ultrafiltration [115] and gel filtration [116] are more
efficient than dialysis for large scale apoflavoprotein pre-
paration. This is especially important when using extreme
conditions. Apoglucose oxidase, for instance, can be
prepared by acidification to pH 1.4–1.8, followed by gel
filtration in the presence of 30% glycerol [55,62,117]. Based
on far-UV circular dichroism data it was suggested that
under these conditions, the apoprotein retains a compact

Old yellow enzyme [93] p-Hydroxybenzoate hydroxylase [174]
D
-Amino-acid oxidase [104] Covalent chromatography
Cytochrome b5 reductase [98] p-Hydroxybenzoate hydroxylase [114,239]
Oxynitrilase [99] Immobilized metal affinity chromatography
Xanthine oxidase [130] NifL PAS domain [69]
Gel filtration
Glucose oxidase [117]
Carbon monooxide dehydrogenase [137]
Hydroquinone hydroxylase [116]
Ultrafiltration
Cytochrome P450 reductase [115]
4230 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Another conventional procedure of apoprotein prepar-
ation is to partially unfold the holoprotein with guani-
dinium hydrochloride [121,122] or urea [21,123,124].
A disadvantage of this method is that one needs to find
conditions in which the partially unfolded apoprotein is
capable of refolding. Circular dichroism spectroscopy
[46,118,125–127] and fluorescence spectroscopy [127–129]
are useful here to probe the folding behaviour of the protein
of interest.
For many metalloflavoproteins, most of the conventional
apoflavoprotein preparation procedures cause extensive
denaturation. A likely explanation for this is that deflavin-
ation and reconstitution of flavoproteins is difficult if the
quaternary structure is more complex, i.e. when the protein
contains more cofactors and/or subunits. The molybdo-
iron-sulfur flavoprotein xanthine oxidase [130,131] can be
deflavinated by dialysis at physiological pH in the presence

especially when large amounts of apoprotein are required
(affinity-based) chromatographic procedures are the meth-
ods of choice. One of the advantages of these methods is
that on-column protein aggregation is unlikely to occur, as
each molecule is at a relatively large distance from its
neighbours. Particularly during partial unfolding, this helps
stabilizing the apoform of the protein, before flavin
reconstitution.
Ion-exchange chromatography
Many flavoproteins can be reversibly adsorbed to an ion-
exchange support. However, for successful on-column
flavin removal, conditions are needed where the protein
still interacts with the ion-exchanger (low ionic strength) but
not with the flavin (low pH). This concept was first worked
out for the riboflavin-binding proteins from chicken egg
white [140,141] and chicken egg yolk [142,143]. The holo
forms of these carrier proteins can be separated in the apo
forms and free riboflavin by cation-exchange chromatogra-
phy at pH 3.7 [144]. At this pH, riboflavin is released from
the column whereas the apoprotein remains tightly bound.
The apoprotein can subsequently be released from the
column by raising the pH and ionic strength of the elution
buffer.
Unlike most other flavoproteins, aporiboflavin-binding
protein interacts strongly with a large number of flavin
derivatives but not with FMN or FAD [141]. The structure
of chicken egg white riboflavin-binding protein is rather
unusual [145]. Besides from an N-terminal ligand-binding
domain that is strongly conditioned by nine disulfide cross-
links, it contains a flexible phosphorylated motif with nine

(HIC) [150]. This method makes use of the fact that many
flavoproteins bind to phenyl agarose at neutral pH in the
presence of 1
M
ammonium sulfate. Entropy is the driving
force in this process. After immobilization, the flavin can be
removed by the addition of high concentrations of potas-
sium bromide and/or lowering the pH of the elution buffer.
The HIC method has been successfully applied for a
number of flavoproteins [150,151], sometimes with slight
modifications, to get optimal results [37,60,152].
The HIC method for preparing apoflavoproteins works
very well for prokaryotic and eukaryotic disulfide reduc-
tases, including lipoamide dehydrogenase, glutathione
reductase, and mercuric reductase, and is preferred over
classical methods [150]. For apolipoamide dehydrogenase it
was shown that the kinetics of holoenzyme reconstitution
are dependent on the source of enzyme [151] and on the type
of flavin [153]. Initial FAD binding to the monomeric
apoprotein results in dichlorophenol-indophenol activity
and quenching of tryptophan fluorescence. Then, dimeriza-
tion occurs as reflected by the lipoamide activity, strongly
increased FAD fluorescence and increased hyperchroism of
Ó FEBS 2003 Flavoprotein resolution and reconstitution (Eur. J. Biochem. 270) 4231
the visible absorption spectrum [151]. For lipoamide
dehydrogenase from A. vinelandii, the conformational sta-
bility of the monomeric apoprotein was compared with that
of the dimeric holoenzyme [128]. Unfolding of the apo-
enzyme in guanidinium hydrochloride follows a simple two-
state mechanism and is fully reversible. However, the

apoprotein preparation. At pH 7.0 and 25 °C, the apopro-
tein is a mixture of dimers and tetramers, and reassociates to
a native-like tetrameric form in the presence of FAD. The
reconstitution with FAD is relatively slow, and is stimulated
in the presence of CoA ligands. Binding of CoA ligands
stimulates tetramerization of the reconstituted holoenzyme
and improves protein stability. This is in agreement with the
crystal structure of butyryl-CoA dehydrogenase [158] which
shows that the inhibitor acetoacetyl-CoA binds in an
extended conformation near the dimer–dimer interface.
Fluorescence/polarization experiments revealed that the
reconstituted protein is somewhat less stable than the native
holoprotein, and that FAD dissociates more easily [150].
Another protein that was successfully deflavinated by the
HIC method is
L
-amino-acid oxidase from the venom of
Crotalus adamanteus, the eastern diamondback rattlesnake
[159].
L
-amino-acid oxidase is a dimeric glycoprotein,
containing one FAD per monomer, that catalyses the
oxidative deamination of
L
-amino acids. The deflavination
method for
L
-amino-acid oxidase [160] is similar to the
original protocol developed for lipoamide dehydrogenase.
The apo form of

flavin affinity. From this it was concluded that glycerol
assists in the rearrangement of the protein towards the
holoprotein conformation, as well as in reducing the solvent
accessibility of the protein hydrophobic core [163].
Hydroxyapatite chromatography
Hydroxyapatite chromatography (HAP) is often used as a
final step in protein purification. For preparation and
reconstitution of apoflavoproteins, HAP has the advantage
that high salt concentrations, which can stimulate apo-
protein formation, are not necessarily a limitation. The
interaction between the protein and hydroxyapatite is
primarily the result of non-specific electrostatic interactions
between the positively charged protein amino groups and
the negatively charged column material [164]. The deflavi-
nation process is influenced by the charge distribution of the
protein, as well as the kind of salt that is used as eluent. The
HAP method of apoflavoprotein preparation was used for a
recombinant form of the flavin-binding subunit of p-cresol
methylhydroxylase from Pseudomonas putida [165] and for
the His61Thr variant of vanillyl-alcohol oxidase (VAO)
from Penicillium simplicissimum [166,167]. Binding the
His61Thr variant to hydroxyapatite appeared to be a very
gentle and efficient method of obtaining the VAO apopro-
tein. Upon washing with 200 m
M
phosphate buffer, the
His61Thr protein remains tightly bound to the column,
whereas the FAD is easily removed. This removal of FAD is
superior to other methods. For instance, when the His61Thr
holoenzyme is gel filtrated in the absence or presence of high

of the column binds to the enzyme, displacing the FAD. The
column is then eluted with high-ionic strength buffer
containing the artificial flavin 6-azido-FAD, which binds
totheproteinanddisplacesthedye.The6-azido-FAD
cofactor can be covalently linked to the protein by
irradiation. Enzyme that has not been photolabeled is
separated from the covalently photolabeled enzyme by
applying the reaction mixture to a Red-A column again. At
low ionic strength, the nonlabeled enzyme binds to the
column material, whereas the photolabeled enzyme passes
directly through the column [77,80].
Covalent chromatography
When partial unfolding of the holoprotein is required to
weaken the protein–flavin interaction, the above mentioned
chromatographic methods for protein deflavination may
not work properly because of the presence of high
concentrations of unfolding agents. Therefore, we intro-
duced the concept of covalent enzyme immobilization for
improving the yield and quality of the apoprotein of PHBH
from P. fluorescens [114].
PHBH from P. fluorescens is a homodimeric FAD-
dependent monooxygenase that contains 5 sulfhydryl groups
per monomer [175]. Cys116 is the only solvent exposed thiol
group, accessible to N-ethylmaleimide and 5,5¢-dithio-bis(2-
nitrobenzoate) (Nbs2) [176]. Using this property, it is
possible to bind the enzyme covalently to a Nbs2–agarose
column [114]. Oxidation [92] or mutation [177] of Cys116
does not influence catalysis but prohibits binding of the
protein to the Nbs2 column. In Fig. 2, the PHBH dimer is
shown together with the solvent accessibility of the Cys116

flavoprotein bound to an IMAC column is in principle able
to withstand rather harsh conditions that can be used to
successfully remove the flavin prosthetic group.
The IMAC method of apoflavoprotein preparation was
developed for the flavin-containing PAS domain of NifL, a
redox-sensing protein from A. vinelandii [180,181]. Deflavi-
nation was achieved on a nickel-nitrilotriacetic acid column
by exploiting the available N-terminal His-tag [69]. Protein-
bound FAD was removed efficiently by washing the column
Fig. 2. 3D-structure of PHBH. The FAD cofactor is shown in green. In the right panel, all amino-acid residues within a distance of 10 A
˚
to the
Cys116 sulfur atom (yellow) are shown. The solvent accessibility of the sulfur atom is indicated with yellow dots, and Cys116 is drawn with
Vanderwaals-radii.
Ó FEBS 2003 Flavoprotein resolution and reconstitution (Eur. J. Biochem. 270) 4233
with KBr and urea. The apoprotein could be eluted from
the column with imidazole, but slowly precipitated after
column release. Therefore, on-column reconstitution was
performed by circulating a solution of 2,4a-
13
C
2
-FAD or
2,4a-
13
C
2
-FMN (Fig. 3).
The reconstituted PAS domain containing a new flavin
cofactor was eluted from the column with imidazole.

mechanism of covalent flavinylation. One of these strategies,
developed for human monoamine oxidase, makes use of a
yeast strain auxotrophic for riboflavin and expression of the
enzyme in this strain in the presence of different riboflavin
analogs [192,193]. Another more generally applied strategy
is based on the replacement of crucial amino-acid residues
by site-directed mutagenesis [194–198]. In short, these
investigations have supported early proposals from model
system studies [199,200] that covalent flavinylation involves
an autocatalytical iminoquinone-methide addition mechan-
ism with flavin binding preceding covalent attachment
[8,39]. In line with this mechanism it was found that the
apoenzyme of monoamine oxidase B, expressed in COS
cells devoid of riboflavin, is correctly inserted into the outer
mitochondrial membrane [201].
For succinate dehydrogenase from yeast it was esta-
blished that flavinylation takes place within the mitochond-
rial matrix after import of the flavoprotein subunit and the
cleavage of a leader peptide [186,202,203]. Moreover,
flavinylation of this iron-sulfur flavoenzyme was enhanced
in the presence of the chaperone protein hsp60 [186].
p-Cresol methylhydroxylase is an 8a-O-tyrosyl-FAD
containing flavocytochrome involved in the anaerobic
microbial degradation of alkylphenols. From individual
expression of the heme- and flavin-binding subunits it was
revealed that the apoflavoprotein component of p-cresol
methylhydroxylase is capable of noncovalently binding
FAD but that the interaction with the heme-containing
subunit is required for the self-catalytic flavinylation reac-
tion [204,205].

[211,212]. From MALDI-TOF MS analysis of proteolytic
digests, it was concluded that in both NqrB and NqrC
subunits, the FMN cofactor is attached by its 5¢-phosphate
moiety to a threonine side chain. In agreement with this, no
covalent flavin was detected in the Thr225Leu mutant of
NqrC from Vibrio cholerae [213]. From sequence compari-
sons it was predicted that this novel type of phosphoester
Fig. 3. Schematic representation of the preparation and reconstitution of
His-tagged apoflavoproteins by immobilization on a nickel-nitrilotri-
acetic acid column (from [69], with permission).
4234 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
binding between FMN and the apoprotein is conserved in
the NADH-quinone oxidoreductase sodium pumping sys-
tems of a number of marine and pathogenic bacteria and
that in some of these systems the target threonine is replaced
by a serine residue [214].
Conclusions and future perspectives
Since the pioneering work of Theorell [93], many methods
have been developed for the (large scale) preparation and
reconstitution of apoflavoproteins. Conventional precipita-
tion methods are rapid but the yield and reconstitutability of
apoprotein may vary dramatically. More recently developed
chromatographic procedures have the advantage that the
apoprotein is stabilized by immobilization, and that large
amounts of apo- or reconstituted flavoproteins can be
obtained.
His-tagged flavoproteins can be purified, deflavinated
and reconstituted on the same IMAC column. Therefore,
the use of IMAC for flavoprotein deflavination and
reconstitution should be further exploited. An interesting

However, the Arg386Leu mutant, in which an active site
arginine is replaced, turned out to be amenable to crystal-
lization and structure elucidation in the active FAD-bound
state [219].
Understanding the specific interaction between flavopro-
teins and their cofactors is also of medical relevance. Since
the earlier finding that a reduced affinity of human
glutathione reductase for FAD due to a mutation can lead
to nonspherocytic haemolytic anaemia [220], several genetic
defects affecting flavin binding have been described. Muta-
tions causing impaired flavin binding have been reported
for, e.g. NADPH-oxidase [221–225], NADH:cytochrome b
5
reductase [226–228], methylenetetrahydrofolate reductase
[229–234], and dihydropyrimidine dehydrogenase [235] and
the consequences at a molecular level are starting to emerge.
Apoptosis-inducing factor is a flavoprotein that can stimu-
late a caspase-independent cell-death pathway required for
early embryonic morphogenesis [236,237]. To gain further
insight into the redox properties of apoptosis-inducing
factor, Lys176 and Glu313, located near the isoalloxazine
ring of FAD, were individually changed into alanine by
site-directed mutagenesis. Both apoptosis-inducing factor
variants appeared to be highly active when assayed in the
presence of excess FAD. However, during purification
the Lys176Ala and Glu313Ala mutant enzymes easily lost
the flavin cofactor, yielding the corresponding apoproteins
[238]. This again demonstrates that changing a specific
amino-acid residue can considerably influence the strength
of flavin binding.

catalyzed reactions. Eur. J. Biochem. 181, 1–17.
7. Ballou, D.P., Williams, C.H. & Coon, M.J. (2002) Vincent
Massey (1926–2002). Trends Biochem. Sci. 27, 641–642.
8. Mewies, M., McIntire, W.S. & Scrutton, N.S. (1998) Covalent
attachment of flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN) to enzymes: The current state of affairs.
Protein Sci. 7, 7–20.
9. Mathews, F.S. (1991) New flavoenzymes. Curr. Opin. Struct.
Biol. 1, 954–967.
10. Fraaije, M.W. & Mattevi, A. (2000) Flavoenzymes: diverse
catalysts with recurrent features. Trends Biochem. Sci. 25,
126–132.
11. Dym, O. & Eisenberg, D. (2001) Sequence-structure analysis of
FAD-containing proteins. Protein Sci. 10, 1712–1728.
12. Lee, Y.H., Nadaraia, S., Gu, D., Becker, D.F. & Tanner, J.J.
(2003) Structure of the proline dehydrogenase domain of the
multifunctional PutA flavoprotein. Nat. Struct. Biol. 10, 109–
114.
13. Eggink,G.,Engel,H.,Vriend,G.,Terpstra,P.&Witholt,B.
(1990) Rubredoxin reductase of Pseudomonas oleovorans. Struc-
tural relationship to other flavoprotein oxidoreductases based
on one NAD and two FAD fingerprints. J. Mol. Biol. 212,
135–142.
14. Vallon, O. (2000) New sequence motifs in flavoproteins: evidence
for common ancestry and tools to predict structure. Proteins 38,
95–114.
15. Swindells, M.B. (1993) Classification of doubly wound nucleo-
tide binding topologies using automated loop searches. Protein
Sci. 2, 2146–2153.
16. Eppink, M.H.M., Schreuder, H.A. & van Berkel, W.J.H. (1997)

T.W.,Daff,S.,Miles,C.S.,Chapman,S.K.,Lysek,D.A.,Moser,
C.C., Page, C.C. & Dutton, P.L. (2002) P450 BM3: the very
model of a modern flavocytochrome. Trends Biochem. Sci. 27,
250–257.
25. Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D.,
Watkins,D.,Heng,H.H.,Rommens,J.M.,Scherer,S.W.,
Rosenblatt, D.S. & Gravel, R.A. (1998) Cloning and mapping
of a cDNA for methionine synthase reductase, a flavoprotein
defective in patients with homocystinuria. Proc. Natl Acad. Sci.
USA 95, 3059–3064.
26. Olteanu, H. & Banerjee, R. (2001) Human methionine synthase
reductase, a soluble P-450 reductase-like dual flavoprotein, is
sufficient for NADPH-dependent methionine synthase activa-
tion. J. Biol. Chem. 276, 35558–35563.
27. Eschenbrenner, M., Coves, J. & Fontecave, M. (1995) NADPH-
sulfite reductase flavoprotein from Escherichia coli: contribution
to the flavin content and subunit interaction. FEBS Lett. 374,
82–84.
28. Eschenbrenner, M., Coves, J. & Fontecave, M. (1995) The flavin
reductase activity of the flavoprotein component of sulfite
reductase from Escherichia coli. A new model for the protein
structure. J. Biol. Chem. 270, 20550–20555.
29. Ostrowski, J., Barber, M.J., Rueger, D.C., Miller, B.E., Siegel,
L.M. & Kredich, N.M. (1989) Characterization of the flavo-
protein moieties of NADPH-sulfite reductase from Salmonella
typhimurium and Escherichia coli. J. Biol. Chem. 264, 15796–
15808.
30. Zeghouf, M., Fontecave, M., Macherel, D. & Coves, J. (1998)
The flavoprotein component of the Escherichia coli sulfite
reductase: expression, purification, and spectral and catalytic

ller, F., ed.), pp. 261–274.
CRC Press Inc, Boca Raton, FL.
37. Lederer, F., Ru
¨
terjans, H. & Fleischmann, G. (1999) Flavopro-
tein resolution and reconstitution. In Flavoprotein Protocols
(Chapman, S.K. & Reid, G.A., eds), pp. 149–155. Humana Press
Inc, Totower, New Jersey.
38. Ghisla, S. & Massey, V. (1986) New flavins for old: artificial
flavins as active site probes of flavoproteins. Biochem. J. 239,
1–12.
39. Edmondson, D.E. & Newton-Vinson, P. (2001) The covalent
FAD of monoamine oxidase: structural and functional role and
mechanism of the flavinylation reaction. Antioxidant Redox
Signal. 3, 789–806.
40. Mateo, P.L. & Sturtevant, J.M. (1977) Thermodynamics of the
binding of flavin adenine dinucleotide to
D
-amino acid oxidase.
Biosystems 8, 247–253.
4236 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
41. Sturtevant, J.M. & Mateo, P.L. (1978) Proposed temperature-
dependent conformational transition in
D
-amino acid oxidase: a
differential scanning microcalorimetric study. Proc. Natl Acad.
Sci. USA 75, 2584–2587.
42. Lostao,A.,ElHarrous,M.,Daoudi,F.,Romero,A.,Paro-
dy-Morreale, A. & Sancho, J. (2000) Dissecting the energetics of
the apoflavodoxin-FMN complex. J. Biol. Chem. 275, 9518–

actions and the redox properties of the Shethna flavoprotein.
Biochemistry 10, 133–145.
51. Smith, W.W., Burnett, R.M., Darling, G.D. & Ludwig, M.L.
(1977) Structure of the semiquinone form of flavodoxin from
Clostridum MP. Extension of 1.8 A
˚
resolution and some com-
parisons with the oxidized state. J. Mol. Biol. 117, 195–225.
52. Massey, V., Ghisla, S. & Moore, E.G. (1979) 8-Mercaptoflavins
as active site probes of flavoenzymes. J. Biol. Chem. 254, 9640–
9650.
53. Manstein, D.J., Massey, V., Ghisla, S. & Pai, E.F. (1988) Ste-
reochemistry and accessibility of prosthetic groups in flavopro-
teins. Biochemistry 27, 2300–2305.
54. van Berkel, W.J.H., Eppink, M.H.M. & Schreuder, H.A. (1994)
Crystal structure of p-hydroxybenzoate hydroxylase recon-
stituted with the modified FAD present in alcohol oxidase from
methylotrophic yeasts: Evidence for an arabinoflavin. Protein
Sci. 3, 2245–2253.
55. Riklin, A., Katz, E., Willner, I., Stocker, A. & Bu
¨
ckmann, A.F.
(1995) Improving enzyme-electrode contacts by redox modifica-
tion of cofactors. Nature 376, 672–675.
56. Murthy, Y.V.S.N. & Massey, V. (1997) Synthesis and applica-
tions of flavin analogs as active site probes of flavoproteins.
Methods Enzymol. 280, 436–460.
57. Moonen, C.T. & Mu
¨
ller, F. (1983) On the mobility of riboflavin

5156–5167.
61. Salomon, M., Eisenreich, W., Duerr, H., Schleicher, E., Knieb,
E.,Massey,V.,Ruediger,W.,Mu
¨
ller, F., Bacher, A. & Richter,
G. (2001) An optomechanical transducer in the blue light
receptor phototropin from Avena sativa. Proc. Natl Acad. Sci.
USA 98, 12357–12361.
62. Sanner,C.,Macheroux,P.,Ru
¨
terjans, H., Mu
¨
ller, F. & Bacher,
A. (1991)
15
Nand
13
C-NMR investigations of glucose oxidase
from Aspergillus niger. Eur. J. Biochem. 196, 663–672.
63. Stockman,B.J.,Westler,W.M.,Mooberry,E.S.&Markley,J.L.
(1998) Flavodoxin from Anabaena 7120: uniform nitrogen-15
enrichment and hydrogen-1, nitrogen-15, and phosphorous-31
NMR investigations of the flavin mononucleotide binding site in
the reduced and oxidized state. Biochemistry 27, 136–142.
64. Vervoort,J.,vanBerkel,W.J.H.,Mu
¨
ller, F. & Moonen, C.T.W.
(1991) NMR studies on p-hydroxybenzoate hydroxylase from
Pseudomonas fluorescens and salicylate hydroxylase from Pseu-
domonas putida. Eur.J.Biochem.200, 731–738.

68. Miura, R. & Miyake, Y. (1987)
13
C-NMR studies of porcine
kidney
D
-amino acid oxidase reconstituted with
13
C-enriched
flavin adenine dinucleotide. Effects of competitive inhibitors.
J. Biochem. (Tokyo) 101, 581–589.
69. Hefti, M.H., Milder, F.J., Boeren, S., Vervoort, J. & van Berkel,
W.J.H. (2003) A His-tag based immobilization method for the
preparation and reconstitution of apoflavoproteins. Biochim.
Biophys. Acta 1619, 139–143.
70. Mu
¨
ller, F. (1991) Nuclear magnetic resonance studies of flavo-
proteins. In Chemistry and Biochemistry of Flavoenzymes
(Mu
¨
ller, F., ed.), pp. 557–595. CRC Press Inc, Boca Raton, FL.
71. Schopfer, L.M., Massey, V. & Claiborne, A. (1981) Active site
probes of flavoproteins. Determination of the solvent accessi-
bility of the flavin position 8 for a series of flavoproteins. J. Biol.
Chem. 256, 7329–7337.
72. Claiborne, A., Massey, V., Fitzpatrick, P.F. & Schopfer, L.M.
(1982) 2-Thioflavins as active site probes of flavoproteins. J. Biol.
Chem. 257, 174–182.
73.Massey,V.,Claiborne,A.,Biemann,M.&Ghisla,S.(1984)
4-Thioflavins as active site probes of flavoproteins. J. Biol. Chem.

relationship data for lactate oxidase. Proc. Natl Acad. Sci. USA
97, 2480–2485.
83. Manstein, D.J., Pai, E.F., Schopfer, L.M. & Massey, V. (1986)
Absolute stereochemistry of flavins in enzyme-catalyzed reac-
tions. Biochemistry 25, 6807–6816.
84. Bross, P., Jensen, T.G., Andresen, B.S., Kjeldsen, M., Nandy, A.,
Kolvraa, S., Ghisla, S., Rasched, I., Bolund, L. & Gregersen, N.
(1994) Characterization of wild-type human medium-chain
acyl-CoA dehydrogenase (MCAD) and mutant enzymes present
in MCAD-deficient patients by two-dimensional gel electro-
phoresis: evidence for post-translational modification of the
enzyme. Biochem. Medical Metab. Biol. 52, 36–44.
85. Murthy, Y.V. & Massey, V. (1995) Chemical modification of the
N-10 ribityl side chain of flavins. Effects on properties of flavo-
protein disulfide oxidoreductases. J. Biol. Chem. 270, 28586–
28594.
86. Palfey, B.A., Murthy, Y.V. & Massey, V. (2003) Altered balance
of half-reactions in p-hydroxybenzoate hydroxylase caused by
substituting the 2¢-carbon of FAD with fluorine. J. Biol. Chem.
278, 22210–22216.
87. Miller, S.M. (1995) 2¢-fluoro-2¢-deoxy-
D
-arabinoflavin: charac-
terization of a novel flavin and its effects on the formation
and stability of two-electron-reduced mercuric ion reductase.
Biochemistry 34, 13066–13073.
88. Thorpe, C. & Kim, J.J. (1995) Structure and mechanism of action
of the acyl-CoA dehydrogenases. FASEB J. 9, 718–725.
89. Engst, S., Vock, P., Wang, M., Kim, J.J. & Ghisla, S. (1999)
Mechanism of activation of acyl-CoA substrates by medium

tischen Gruppe der
D
-Aminosa
¨
ureoxydase. Biochemische Z. 298,
150–168.
95. Haas, E. (1938) Isolierung eines neuen gelben Ferments. Bio-
chemische Z. 298, 378–390.
96. Swoboda, B.E. (1969) The relationship between molecular con-
formation and the binding of flavin-adenine dinucleotide in
glucose oxidase. Biochim. Biophys. Acta 175, 365–379.
97. Kalse, J.F. & Veeger, C. (1968) Relation between conformations
and activities of lipoamide dehydrogenase. I. Relation between
diaphorase and lipoamide dehydrogenase activities upon binding
of FAD by the apoenzyme, Biochim. Biophys. Acta 159,
244–256.
98. Strittmatter, P. (1961) The nature of the flavin binding in
microsomal cytochrome b
5
reductase. J. Biol. Chem. 236, 2329–
2335.
99. Schuman Jorns, M. (1979) Mechanism of catalysis by the flavo-
enzyme oxynitrilase. J. Biol. Chem. 254, 12145–12152.
100. Mayer, E.J. & Thorpe, C. (1981) A method for resolution of
general acyl-coenzyme A dehydrogenase apoprotein. Anal Bio-
chem. 116, 227–229.
101. Brock, B.J. & Gold, M.H. (1996) 1,4-Benzoquinone reductase
from basidiomycete Phanerochaete chrysosporium:spectraland
kinetic analysis. Arch. Biochem. Biphys. 331, 31–40.
102. Mayhew, S.G. (1971) Studies on flavin binding in flavodoxins.

hydroxylase. Biochemistry 22, 580–584.
110. Radjendirane, V., Bhat, M.A. & Vaidyanathan, C.S. (1991)
Affinity purification and characterization of 2,4-dichlorophenol
hydroxylase from Pseudomonas cepacia. Arch. Biochem. Biophys.
288, 169–176.
111. Tu, S.C. & McCormick, D.B. (1974) Conformation of porcine
D
-amino acid oxidase as studied by protein fluorescence and
optical rotatory dispersion. Biochemistry 13, 893–899.
112. Zlateva, T., Boteva, R., Filippi, B., Veenhuis, M. & van der Klei,
I.J. (2001) Deflavination of flavo-oxidases by nucleophilic
reagents. Biochim. Biophys. Acta 1548, 213–219.
4238 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
113. Pollegioni, L., Iametti, S., Fessas, D., Caldinelli, L., Piubelli, L.,
Barbiroli, A., Pilone, M.S. & Bonomi, F. (2003) Contribution of
the dimeric state to the thermal stability of the flavoprotein
D
-amino acid oxidase. Protein Sci. 12, 1018–1029.
114. Mu
¨
ller, F. & van Berkel, W.J.H. (1982) A study on p-hydro-
xybenzoate hydroxylase from Pseudomonas fluorescens.Acon-
venient method of preparation and some properties of the
apoenzyme. Eur.J.Biochem.128, 21–27.
115. Bonants, P.J., Mu
¨
ller, F., Vervoort, J. & Edmondson, D.E.
(1990) A
31
P-nuclear-magnetic-resonance study of NADPH-

122. Munro, A.W., Coggins, J.R., Lindsay, J.G., Kelly, S. & Price,
N.C. (1996) Deflavination of cytochrome P450 BM3 by treat-
ment with guanidinium chloride. Biochem. Soc. Trans. 24, 19S.
123. Wang, L.H., Tu, S.C. & Lusk, R.C. (1984) Apoenzyme of
Pseudomonas cepacia salicylate hydroxylase. Preparation, fluor-
escence property, and nature of flavin binding. J. Biol. Chem.
259, 1136–1142.
124. Choong, Y.S., Shepherd, M.G. & Sullivan, P.A. (1975) Pre-
paration of the lactate oxidase apoenzyme and studies on the
binding of flavin mononucleotide to the apoenzyme. Biochem.
J. 145, 37–45.
125. Maeda,M.,Hamada,D.,Hoshino,M.,Onda,Y.,Hase,T.&
Goto, Y. (2002) Partially folded structure of flavin adenine
dinucleotide-depleted ferredoxin-NADP
+
reductase with resid-
ual NADP
+
binding domain. J. Biol. Chem. 277, 17101–17107.
126. Munro, A.W., Kelly, S.M. & Price, N.C. (1999) Circular
dichroism studies of flavoproteins. Methods Mol. Biol. 131,111–
123.
127. van Mierlo, C.P.M., van Dongen, W.M.A.M., Vergeldt, F., van
Berkel, W.J.H. & Steensma, E. (1998) The equilibrium unfolding
of Azotobacter vinelandii apoflavodoxin II occurs via a relatively
stable folding intermediate. Protein Sci. 7, 2331–2344.
128. van Berkel, W.J.H., Regelink, A.G., Beintema, J.J. & de Kok, A.
(1991) The conformational stability of the redox states of lipo-
amide dehydrogenase from Azotobacter vinelandii. Eur. J. Bio-
chem. 202, 1049–1055.

Escherichia coli. J. Biol. Chem. 275, 1864–1872.
138. van Mierlo, C.P.M., Vervoort, J., Mu
¨
ller, F. & Bacher, A. (1990)
Atwo-dimensional
1
HNMRstudyonMegasphaera elsdenii
flavodoxin in the reduced state. Sequential assignments. Eur.
J. Biochem. 187, 521–541.
139. van Mierlo, C.P.M., van der Sanden, B.P., van Woensel, P.,
Mu
¨
ller, F. & Vervoort, J. (1990) A two-dimensional
1
H-NMR
study on Megasphaera elsdenii flavodoxin in the oxidized state
and some comparisons with the two-electron-reduced state. Eur.
J. Biochem. 194, 199–216.
140. Farrell, H.M Jr, Mallette, M.F., Buss, E.G. & Clagett, C.O.
(1969) The nature of the biochemical lesion in avian renal ribo-
flavinuria. 3. The isolation and characterization of the riboflavin-
binding protein from egg albumin. Biochim. Biophys. Acta 194,
433–442.
141. Becvar, J. & Palmer, G. (1982) The binding of flavin derivatives
to the riboflavin-binding protein of egg white. A kinetic and
thermodynamic study. J. Biol. Chem. 257, 5607–5617.
142. Matsui, K., Sugimoto, K. & Kasai, S. (1982) Thermodynamics of
association of egg yolk riboflavin binding protein with 8-sub-
stituted riboflavins. Comparison with the egg white protein.
J. Biochem. (Tokyo) 91, 1357–1362.

Large-scale preparation and reconstitution of apo-flavoproteins
with special reference to butyryl-CoA dehydrogenase from
Megasphaera elsdenii. Eur. J. Biochem. 178, 197–208.
151. van Berkel, W.J.H., Benen, J.A.E. & Snoek, M.C. (1991) On the
FAD-induced dimerization of apo-lipoamide dehydrogenase
from Azotobacter vinelandii and Pseudomonas fluorescens:
Kinetics of reconstitution. Eur.J.Biochem.197, 769–780.
152. Schuman Jorns, M., Wang, B., Jordan, S.P. & Chanderkar, L.P.
(1990) Chromophore function and interaction in Escherichia coli
DNA photolyase: Reconstitution of the apoenzyme with pterin
and/or flavin derivatives. Biochemistry 29, 552–561.
153. de Kok, A. & van Berkel, W.J.H. (1996) Lipoamide dehydro-
genase. In Alpha-Keto Acid Dehydrogenase Complexes (Patel,
M.S., Roche, T.E. & Harris, R.A., eds), pp. 53–70. Birkha
¨
user-
Verlag, Basel.
154. Benen, J., van Berkel, W.J.H., Dieteren, N., Arscott, D.,
Williams,CJr,Veeger,C.&deKok,A.(1992)Lipoamide
dehydrogenase from Azotobacter vinelandii: site-directed muta-
genesis of the His450-Glu455 diad. Kinetics of wild-type and
mutated enzymes. Eur. J. Biochem. 207, 487–497.
155. Benen, J., van Berkel, W.J.H., Veeger, C. & de Kok, A. (1992)
Lipoamide dehydrogenase from Azotobacter vinelandii. The role
of the C-terminus in catalysis and dimer stabilization. Eur.
J. Biochem. 207, 499–505.
156. Mattevi, A., Obmolova, G., Kalk, K.H., van Berkel, W.J.H. &
Hol, W.G. (1993) Three-dimensional structure of lipoamide
dehydrogenase from Pseudomonas fluorescens at 2.8 A
˚

(Tokyo) 109, 171–177.
163. Raibekas, A.A. & Massey, V. (1997) Glycerol-assisted restorative
adjustment of flavoenzyme conformation perturbed by site-
directed mutagenesis. J. Biol. Chem. 272, 22248–22252.
164. Gorbunoff, M.J. (1984) The interaction of proteins with
hydroxyapatite. Anal Biochem. 136, 425–445.
165. Efimov, I., Cronin, C.N. & McIntire, W.S. (2001) Effects of
noncovalent and covalent FAD binding on the redox and
catalytic properties of p-cresol methylhydroxylase. Biochemistry
40, 2155–2166.
166. Fraaije, M.W., van den Heuvel, R.H.H., van Berkel, W.J.H. &
Mattevi, A. (2000) Structural analysis of flavinylation in vanillyl-
alcohol oxidase. J. Biol. Chem. 275, 38654–38658.
167. Tahallah, N., van den Heuvel, R.H., van den Berg, W.A.M.,
Maier, C.S., van Berkel, W.J.H. & Heck, A.J.R. (2002) Cofactor-
dependent assembly of the flavoenzyme vanillyl-alcohol oxidase.
J. Biol. Chem. 277, 36425–36432.
168. Dean, P.D. & Watson, D.H. (1979) Protein purification using
immobilised triazine dyes. J. Chromatog. 165, 301–319.
169. Rossman, M.G., Liljas, A., Bra
¨
nde
´
n, C I. & Banaszak, L.J.
(1975) Evolutionary and structural relationships among dehy-
drogenases. Enzymes 11, 61–102.
170. Wierenga, R.K., Drenth, J. & Schulz, G.E. (1983) Comparison of
the three-dimensional protein and nucleotide structure of the
FAD-binding domain of p-hydroxybenzoate hydroxylase with
the FAD- as well as NADPH-binding domains of glutathione

¨
ller,F.,Jekel,P.A.&
Beintema, J.J. (1984) Chemical modification of sulfhydryl groups
in p-hydroxybenzoate hydroxylase from Pseudomonas fluor-
escens. Involvement in catalysis and assignment in the sequence.
Eur. J. Biochem. 145, 245–256.
177. Eschrich, K., van Berkel, W.J.H., Westphal, A.H., de Kok, A.,
Mattevi, A., Obmolova, G., Kalk, K.H. & Hol, W.G. (1990)
Engineering of microheterogeneity-resistant p-hydroxybenzoate
hydroxylase from Pseudomonas fluorescens. FEBS Lett. 277,
197–199.
178. Ortiz-Maldonado, M., Aeschliman, S.M., Ballou, D.P. &
Massey, V. (2001) Synergistic interactions of multiple mutations
on catalysis during the hydroxylation reaction of p-hydro-
xybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp,
and Tyr385Phe mutants reconstituted with 8-Cl- flavin.
Biochemistry 40, 8705–8716.
179. Ortiz-Maldonado, M., Ballou, D.P. & Massey, V. (2001) A rate-
limiting conformational change of the flavin in p-hydro-
xybenzoate hydroxylase is necessary for ligand exchange and
catalysis: studies with 8-mercapto- and 8-hydroxy-flavins.
Biochemistry 40, 1091–1101.
180. Dixon, R. (1998) The oxygen-responsive NIFL-NIFA complex: a
novel two-component regulatory system controlling nitrogenase
4240 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003
synthesis in gamma-proteobacteria. Arch. Microbiol. 169,371–
380.
181. Hefti,M.H.,vanVugt-vanderToorn,C.J.G.,Dixon,R.&
Vervoort, J. (2001) A novel purification method for histidine-
tagged proteins containing a thrombin cleavage site. Anal Bio-

function relationships in flavoenzyme-dependent amine oxi-
dations: a comparison of polyamine oxidase and monoamine
oxidase. J. Biol. Chem. 277, 23973–23976.
191. Kearney, E.B., Salach, J.I., Walker, W.H., Seng, R.L., Kenney,
W., Zeszotek, E. & Singer, T.P. (1971) The covalently-bound
flavin of hepatic monoamine oxidase. 1. Isolation and sequence
of a flavin peptide and evidence for binding at the 8a position.
Eur. J. Biochem. 24, 321–327.
192. Miller, J.R., Guan, N., Hubalek, F. & Edmondson, D.E. (2000)
The FAD binding sites of human liver monoamine oxidases A
and B: investigation of the role of flavin ribityl side chain
hydroxyl groups in the covalent flavinylation reaction and cata-
lytic activities. Biochim. Biophys. Acta 1476, 27–32.
193. Miller, J.R. & Edmondson, D.E. (1999) Structure-activity
relationships in the oxidation of para-substituted benzylamine
analogues by recombinant human liver monoamine oxidase A.
Biochemistry 38, 13670–13683.
194. Mauch, L., Bichler, V. & Brandsch, R. (1990) Lysine can replace
arginine 67 in the mediation of covalent attachment of FAD to
histidine 71 of 6-hydroxy-
D
-nicotine oxidase. J. Biol. Chem. 265,
12761–12762.
195. Brandsch, R. & Bichler, V. (1991) Autoflavinylation of apo6-
hydroxy-
D
-nicotine oxidase. J. Biol. Chem. 266, 19056–19062.
196. Scrutton, N.S., Packman, L.C., Mathews, F.S., Rohlfs, R.J. &
Hille, R. (1994) Assembly of redox centers in the tri-
methylamine dehydrogenase of bacterium W3A1. Properties of

hydroxylase from two strains of Pseudomonas putida. J. Bacter-
iol. 176, 6349–6361.
205.Kim,J.,Fuller,J.H.,Kuusk,V.,Cunane,L.,Chen,Z.W.,
Mathews, F.S. & McIntire, W.S. (1995) The cytochrome subunit
is necessary for covalent FAD attachment to the flavoprotein
subunit of p-cresol methylhydroxylase. J. Biol. Chem. 270,
31202–31209.
206. Mewies, M., Basran, J., Packman, L.C., Hille, R. & Scrutton,
N.S. (1997) Involvement of a flavin iminoquinone methide in the
formation of 6-hydroxyflavin mononucleotide in trimethylamine
dehydrogenase: a rationale for the existence of 8alpha-methyl
and C6-linked covalent flavoproteins. Biochemistry 36, 7162–
7168.
207. Blaut, M., Whittaker, K., Valdovinos, A., Ackrell, B.A.,
Gunsalus, R.P. & Cecchini, G. (1989) Fumarate reductase
mutants of Escherichia coli that lack covalently bound flavin.
J. Biol. Chem. 264, 13599–13604.
208. Ackrell, B.A.C., Johnson, M.K., Gunsalus, R.P. & Cecchini, G.
(1991) Structure and function of succinate dehydrogenase and
fumarate reductase. In Chemistry and Biochemistry of Flavo-
enzymes (Muller, F., ed.), pp. 229–297. CRC Press Inc, Boca
Raton, FL.
209. Fraaije, M.W., van den Heuvel, R.H.H., van Berkel, W.J.H. &
Mattevi, A. (1999) Covalent flavinylation is essential for efficient
redox catalysis in vanillyl-alcohol oxidase. J. Biol. Chem. 274,
35514–35520.
210. Nandigama, R.K. & Edmondson, D.E. (2000) Influence of FAD
structure on its binding and activity with the C406A mutant of
recombinant human liver monoamine oxidase A. J. Biol. Chem.
275, 20527–20532.

(Mu
¨
ller, F., ed.), pp. 401–425. CRC Press Inc, Boca Raton, FL.
217. Piubelli, L., Caldinelli, L., Molla, G., Pilone, M.S. & Pollegioni,
L. (2002) Conversion of the dimeric
D
-amino acid oxidase from
Rhodotorula gracilis to a monomeric form. A rational mutagen-
esis approach. FEBS Lett. 526, 43–48.
218. Mattevi,A.,Tedeschi,G.,Bacchella,L.,Coda,A.,Negri,A.&
Ronchi, S. (1999) Structure of 1-aspartate oxidase: implications
for the succinate dehydrogenase/fumarate reductase oxido-
reductase family. Struct. Fold. Des. 7, 745–756.
219. Bossi, R.T., Negri, A., Tedeschi, G. & Mattevi, A. (2002)
Structure of FAD-bound 1-aspartate oxidase: insight into sub-
strate specificity and catalysis. Biochemistry 41, 3018–3024.
220. Staal, G.E., Helleman, P.W., de Wael, J. & Veeger, C. (1969)
Purification and properties of an abnormal glutathione reductase
from human erythrocytes. Biochim. Biophys. Acta 185, 63–69.
221. Roos, D. & Winterbourn, C.C. (2002) Lethal weapons. Science
296, 669–671.
222.Leusen,J.H.,Meischl,C.,Eppink,M.H.M.,Hilarius,P.M.,
deBoer,M.,Weening,R.S.,Ahlin,A.,Sanders,L.,Gold-
blatt, D., Skopczynska, H., Bernatowska, E., Palmblad, J.,
Verhoeven, A.J., van Berkel, W.J.H. & Roos, D. (2000) Four
novel mutations in the gene encoding gp91-phox of human
NADPH oxidase: consequences for oxidase assembly. Blood 95,
666–673.
223. Aliverti,A.,Bruns,C.M.,Pandini,V.E.,Karplus,P.A.,Vanoni,
M.A.,Curti,B.&Zanetti,G.(1995)Involvementofserine96

aortic lipid deposition. Hum. Mol. Gen. 10, 433–443.
230. Goyette, P., Sumner, J.S., Milos, R., Duncan, A.M., Rosenblatt,
D.S., Matthews, R.G. & Rozen, R. (1994) Human methylene-
tetrahydrofolate reductase: isolation of cDNA, mapping and
mutation identification. Nat. Genet. 7, 195–200.
231. Frosst,P.,Blom,H.J.,Milos,R.,Goyette,P.,Sheppard,C.A.,
Matthews,R.G.,Boers,G.J.,denHeijer,M.,Kluijtmans,L.A.,
van den Heuvel, L.P. et al. (1995) A candidate genetic risk factor
for vascular disease: a common mutation in methylenetetra-
hydrofolate reductase. Nat. Genet. 10, 111–113.
232. Yamada, K., Chen, Z., Rozen, R. & Matthews, R.G. (2001)
Effects of common polymorphisms on the properties of
recombinant human methylenetetrahydrofolate reductase. Proc.
NatlAcad.Sci.USA98, 14853–14858.
233. Guenther, B.D., Sheppard, C.A., Tran, P., Rozen, R., Matthews,
R.G. & Ludwig, M.L. (1999) The structure and properties of
methylenetetrahydrofolate reductase from Escherichia coli sug-
gest how folate ameliorates human hyperhomocysteinemia. Nat.
Struct. Biol. 6, 359–365.
234. Sibani,S.,Leclerc,D.,Weisberg,I.S.,O’Ferrall,E.,Watkins,D.,
Artigas, C., Rosenblatt, D.S. & Rozen, R. (2003) Characteriza-
tion of mutations in severe methylenetetrahydrofolate reductase
deficiency reveals an FAD-responsive mutation. Hum. Mutat. 21,
509–520.
235. vanKuilenburg,A.B.,Dobritzsch,D.,Meinsma,R.,Haasjes,J.,
Waterham, H.R., Nowaczyk, M.J., Maropoulos, G.D., Hein,
G.,Kalhoff,H.,Kirk,J.M.,Baaske,H.,Aukett,A.,Duley,J.A.,
Ward,K.P.,Lindqvist,Y.&vanGennip,A.H.(2002)Novel
disease-causing mutations in the dihydropyrimidine dehydro-
genase gene interpreted by analysis of the three-dimensional