MINIREVIEW
Structural and mechanistic aspects of flavoproteins:
photosynthetic electron transfer from photosystem I
to NADP
+
Milagros Medina
Departamento de Bioquı
´
mica y Biologı
´
a Molecular y Celular and BFIF, Universidad de Zaragoza, Spain
Introduction
Many electron-transfer reactions in biological systems
depend on redox chains that involve flavoproteins [1].
In these chains, questions remain regarding not only
the mechanisms of electron transfer and hydride
transfer, but also the role that flavins might play in
these events. The primary function of photosystem I
(PSI) is to reduce NADP
+
to NADPH, which is then
used in the assimilation of CO
2
[2,3]. In plants, this
occurs via reduction of the soluble [2Fe–2S] ferre-
doxin (Fd) by PSI. Subsequent reduction of NADP
+
by Fd
rd
is catalysed by FAD-containing ferredoxin–
NADP

This minireview covers the research carried out in recent years into differ-
ent aspects of the function of the flavoproteins involved in cyanobacterial
photosynthetic electron transfer from photosystem I to NADP
+
, flavodox-
in and ferredoxin–NADP
+
reductase. Interactions that stabilize protein–
flavin complexes and tailor the midpoint potentials in these proteins, as
well as many details of the binding and electron transfer to protein and
ligand partners, have been revealed. In addition to their role in photosyn-
thesis, flavodoxin and ferredoxin–NADP
+
reductase are ubiquitous fla-
voenzymes that deliver NAD(P)H or low midpoint potential one-electron
donors to redox-based metabolisms in plastids, mitochondria and bacteria.
They are also the basic prototypes for a large family of diflavin electron
transferases with common functional and structural properties. Under-
standing their mechanisms should enable greater comprehension of the
many physiological roles played by flavodoxin and ferredoxin–NADP
+
reductase, either free or as modules in multidomain proteins. Many aspects
of their biochemistry have been extensively characterized using a combina-
tion of site-directed mutagenesis, steady-state and transient kinetics, spec-
troscopy and X-ray crystallography. Despite these considerable advances,
various key features of the structural–function relationship are yet to be
explained in molecular terms. Better knowledge of these systems and their
particular properties may allow us to envisage several interesting applica-
tions of these proteins beyond their physiological functions.
Abbreviations

⁄ Fld
hq
, substitutes for the Fd
ox
⁄ Fd
rd
pair in this
reaction [5,6].
PSI
rd
þ Fld
sq
! PSI + Fld
hq
NADP
þ
þ 2Fld
hq
¡
FNR
NADPH + 2Fld
sq
Two Fld
sq
molecules transfer two electrons from
two PSI molecules to one FNR. FNR becomes fully
reduced through formation of the intermediate,
FNR
sq
, and later transfers both electrons simulta-

.F
X
is coordinated
by cysteines located in both of the large PSI subunits,
PsaA and PsaB, via a loop that also plays a role in the
attachment of PsaC [12]. PsaC, PsaD and PsaE are
located at the cytosolic site (Fig. 1A) [2,7,13–16]. PsaC
carries the terminal F
A
and F
B
clusters. After binding
of the protein carrier to this PSI site, the electron is
A
F
B
F
A
R39
(PsaE)
K106
(PsaD)
K34
(PsaC)
I59
W57
N58
Y94
D146
D90

carrier are indicated. (C) Molecular surface with electrostatic potential of Anabaena Fld (PDB code 1flv) [28]. Detail of residues in the close
FMN environment in the oxidized Fld O-down conformation is shown on the right. FMN is drawn in CPK (balls or sticks), with carbons
shown in orange. Figure 1(A,B) is reproduced from the supplementary material in Gon˜i et al. [48] .
M. Medina Flavoproteins in photosynthetic electron transfer
FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3943
transferred from F
B
to Fld (or Fd), which subse-
quently leaves the PSI site bringing the electron to
FNR.
Flavins: key cofactors in protein
electron transfer reactions
Free flavins stabilize very little of their one-electron
reduced form, the semiquinone, because the midpoint
potential for reduction of the oxidized state to the
semiquinone, E
ox ⁄ sq
, is more negative than that for
reduction of the semiquinone to the hydroquinone,
E
sq ⁄ hq
[17]. Binding of FAD or FMN to the apopro-
tein usually displaces E
ox ⁄ sq
to a less negative value,
whereas E
sq ⁄ hq
shifts to a more negative value, stabiliz-
ing the semiquinone [1,18,19]. This allows flavoproteins
to function as key intermediates at the interface

observed in the AnFld FMN environment is conserved
in Flds across species [38], but specific interactions
around the flavin vary [39–41]. The 90’s loop usually
provides a Tyr stacked against the FMN si-face (Y94
in AnFld) which makes a large contribution to the
midpoint potential [23–25]. The residue from the 50’s
loop that stacks at the re-inner face is commonly a
Trp (W57 in AnFld) [20,28,29,39,42,43], but nonaro-
matic residues (L, H, M or A) have also been found
[29,40,44,45]. Both residues ensure that the flavin is in
an electronegative environment which allows tight
FMN
hq
binding while making formation of its anion
thermodynamically unfavourable [24,25].
In several Flds, rearrangement of the peptide bond
equivalent to 58–59 in AnFld allows a main chain car-
bonyl to flip from an ‘O-down’ conformation to an
‘O-up’ conformation. In the ‘O-up’ conformation, an
H-bond occurs between this carbonyl and N(5)H from
the neutral semiquinone [46,47]. In Anacystis nidulans
Fld, the flip involves breaking a weak H-bond present
in the oxidized state between the FMN N(5) and the
NH of V59, in favour of a stronger H-bond between
the carbonyl of N58 (‘O-up’ conformation) and FMN
N(5)H [41]. The semiquinone states of A. nidulans and
Anabaena Flds are less stable than those from other
species because the semiquinone H-bond with the CO
of Asn is weaker than the bond formed with the sma-
ller Gly, and because of the presence in the oxidized

stacking of a bulky residue at the re-face of the flavin
decreases the electron-spin density in the benzene ring,
whereas an aromatic residue at the si-face increases the
spin density at N(5) and C(6) [56].
Ferredoxin–NADP
+
reductase
The first structure obtained for a photosynthetic
FNR was from spinach (spFNR). spFNR folds in
two domains, one of which presents a noncovalently
bound FAD molecule and the other binds NADP
+
[57,58]. Structures from other species have also been
reported [59–62]. The FAD-binding domain in
AnFNR includes residues 1–138 and is made up of
six antiparallel b strands arranged in two perpendicu-
Flavoproteins in photosynthetic electron transfer M. Medina
3944 FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS
lar b sheets, with a short a helix at the bottom and
another a helix and a long loop that is maintained by
a small two-stranded antiparallel b sheet at the top
(Fig. 1B). The NADP
+
-binding domain includes resi-
dues 139–303 and consists of a core of five parallel
b strands surrounded by seven a helices [62]. FAD is
bound outside the antiparallel b barrel, and its isoal-
loxazine lies between two tyrosines, Y79 and the C-
terminal Y303 in AnFNR.
The two one-electron midpoint potentials of the fla-

complexation makes electron transfer thermodynami-
cally more favourable [70]. Similarly, the midpoint
potential for reduction of NADP
+
in complex with
FNR is 40 mV less negative than that of the free
NADP
+
⁄ NADPH pair [66,71]. Assignment of hyper-
fine couplings to nuclei of the isoalloxazine semi-
quinone have also been reported for AnFNR
sq
and
pFNR
sq
[72]. These studies indicated that the net effect
of the C-terminal Tyr is withdrawal of electron density
from the benzene ring towards the pyrazine ring, plac-
ing the accepted electron nearer to a site where it can
best be neutralized by protonation – the N5 position.
Electron transfer from PSI to
flavodoxin
Fd and Fld differ in size and in the chemical nature of
their redox cofactors. There is no sequence homology
between them, but structural alignment based on their
surface electrostatic potentials shows cofactor superpo-
sition in the region where both proteins accumulate
the negative end of their molecular dipole moments
[73]. Their biding site on PSI was analysed by studying
the kinetic behaviour of site-directed mutants and by

⁄ Fld
hq
pair is involved in shut-
tling electrons between PSI and FNR, a physiological
role for the Fld
ox
⁄ Fld
sq
pair cannot be precluded
[7,14]. Reduction of AnFld
ox
to the semiquinone state
by PSI has been a useful model with which to ana-
lyse the interaction forces and electron transfer
parameters involved in the physiological reaction [48].
Wild-type AnFld forms a transient PSI:Fld
ox
complex
prior to electron transfer [87]. Site-directed mutagene-
sis has been used to find the role of specific AnFld
side chains in the interaction and electron transfer
with PSI [53,84,88–90]. Many of these Fld mutants
(T12V, E16Q, T56G, W57 replaced by K, R, F, L, A
and Y, I59 replaced by A and K, Y94 replaced by A
and F, N97K, I59A ⁄ I92A and I59E ⁄ I92E) accept
electrons from PSI following transient complex for-
mation [53,87,90,91]. For some (T12V, W57Y and
Y94F), k
et
was lowered considerably, suggesting that

than those obtained for the wild-type in all the
PSI ⁄ Fld ratios assayed [88]. These effects are not
related to a change in Fld midpoint potentials. They
might be interpreted as being caused by a modification
in the accessibility of the flavin, but more generally
can be explained by a conformational change in the
orientation of the interacting PSI and Fld surfaces,
leading to a smaller edge-to-edge distance between F
B
and the flavin ring [48]. However, for other mutations,
rates at a given concentration are lower than the corre-
sponding rate for wild-type Fld. Because, in general,
the introduced mutations are not in the direct isoall-
oxazine coordination, this is unlikely to be because of
differences in the structural FMN environment, but
rather because the orientation between the protein
dipoles is not optimal for electron transfer or because
of a change in the electrostatic potential of the protein
[48].
In conclusion, subtle changes in the isoalloxazine
environment influence Fld binding ability and modu-
late the electron-exchange process by producing differ-
ent orientations and distances between redox centres.
Observations indicate that these side chains contribute
to the orientation of AnFld on the PSI, producing a
wild-type complex that is not the most optimal for
electron transfer. Mutation of these residues changes
Fld surface topology, and the module and orientation
of the molecular dipole, contributing to their altered
behaviour. Mutational studies on I59 and I92 AnFld

the individual residues do not contribute equally to
complex formation with both partners. This was
the case for R16, K72, and particularly K75
[65,89,100,107]. In addition, K138 and R264 in the
NADP
+
-binding domain of AnFNR are more impor-
tant in establishing interactions with AnFld than with
AnFd [100,108]. Moreover, although removal of the
E139 AnFNR negative charge has a deleterious effect
on electron transfer reactions with AnFd, it appears to
enhance electron transfer with AnFld [109]. Electron
transfer with Fld is severely diminished upon the intro-
duction of negatively charged side chains at L76, L78
and V136 in AnFNR [89]. Therefore, these nonpolar
residues participate in the establishment of interactions
with both AnFld and AnFd. With this in mind, it was
expected that one or more negatively charged or
hydrophobic residues on the Fld surface would interact
with some of the above specified residues on FNR.
A number of AnFld variants containing replace-
ments, either at the putative interaction surface with
FNR or in the FMN environment, have been analy-
sed. None of the E16, E20, T56, I59, E61, D65, I92,
Y94, D96 and N97 positions is key, but they do con-
tribute cooperatively to the orientation and strengthen-
ing of the FNR:Fld complexes [53,88]. Simultaneous
replacement of I59 and I92 indicated that they are not
involved in crucial specific interactions [53,89]. T12,
W57 and N58 seem to be more important in the inter-

Fd and Fld, each individual residue does not partici-
pate to the same extent in interactions with each of the
partners [100]. This is in agreement with the fact that,
although multiple chemical modifications produced
Flds less suitable for electron transfer [111], site-direc-
ted mutagenesis has not revealed any residues critical
for the interaction with FNR [53,88–90]. Replacement
of the few Fld positions, T12, W57, N58 and Y94,
with a high interface propensity produced opposing
effects: some Fld:FNR complexes can be either weaker
or stronger and less optimal for electron transfer than
those with wild-type Fld, but others can appear more
optimal for a particular electron transfer process.
Docking suggests that wild-type Fld could adopt
different orientations on the FNR surface without sig-
nificantly altering the distance between the methyl
groups of FAD and FMN (Fig. 2A). This might
explain why subtle changes in the Fld still produce
functional complexes. Moreover, the enhanced or
hindered reactivity can also be explained if there is a
single orientation of Fld in the complex that is
retained and changes either the overall interaction or
the electron transfer parameters. Recent analysis of
multiple charge-reversal mutations on the Fld surface
concluded that interactions do not rely on a precise
complementary surface in the reacting molecules. In
E301
L263
R264
Y79

determinants of the efficient interaction between
Fld and its counterparts
Some mutations appear to favour single orientations
which improve the association and electron transfer
with a particular partner and native Fld complexes are
not the most optimal for electron transfer. Such obser-
vation, in agreement with docking analysis [110], sug-
gests that the flavin atoms might be mainly involved in
the interaction and be solely responsible for electron
transfer. Therefore, subtle changes in the isoalloxazine
environment not only influence Fld-binding abilities,
but also modulate the electron transfer process by pro-
ducing different orientations and distances between the
redox centres. This further confirms that Fld interacts
with different structural partners through nonspecific
interactions, which in turn decrease the potential effi-
ciency that could be achieved if unique and more
favourable orientations were produced with a reduced
number of partners. During Fld-dependent photosyn-
thetic electron transfer, the Fld molecule must move
from its docking site in PSI to that in FNR. In vivo,
the formation of transient complexes of Fld with PSI
and FNR is useful, but not critical, during this process
to promote electron transfer and avoid the reduction
of oxygen by the donor centres [7,8,14,48,53]. Thus,
electrostatic alignment appears to be one of the major
determinants of the orientation of Fld on the partner
surface. The fact that simultaneous replacement on the
Fld surface did not hinder or enhance processes with
PSI and FNR also suggests a different interaction

(NMN) is proposed to bind for hydride transfer [114–
117,119]. S223 and Y235 at the AnFNR 2¢P-AMP site
are critical in determining the specificity and efficient
coenzyme orientation [99,115,121]. The 155–160 and
261–268 loops, which accommodate the coenzyme
pyrophosphate portion, also confer specificity and the
volume of residues in the latter loop fine-tunes FNR
catalytic efficiency [114,116,119,120]. R100 (K166 in
spFNR), situated at the FAD-binding domain, allows
its guanidinium group to H-bond to the NADP
+
pyrophosphate, providing the necessary flexibility to
address the NMN moiety of NADP
+
towards the
active site [99,108,114]. Finally, the Tyr at the si -face
contributes to the correct positioning of the substrate
NADP
+
[68], whereas the C-terminal Tyr at the
re-face is surely critical for modulating NADP
+
⁄ H
biding affinity and selectivity [117,118,122–126].
Structural studies have allowed us to postulate a
stepwise mechanism in which the nucleotide must bind
to a bipartite site [59,62,114,117,127]. The first stage is
recognition of the 2¢P-AMP moiety [62]. The interme-
diate state represents a narrowing of the cavity to
match the adenine and the pyrophosphate, whereas the

first observed process is related to formation of the
FNR
hq
–NADP
+
charge-transfer complex (CTC-2) via
an intermediate Michaelis–Menten complex (MC-2),
followed by hydride transfer to produce an equilibrium
mixture of the CTC-2 and FNR
ox
–NADPH (CTC-1)
CTCs. Both CTCs are also detected for the reverse
reaction, although the mechanism has some differ-
ences. Spectroscopic properties for these CTCs and
their hydride transfer rates for interconversion have
been estimated [66,121]. In AnFNR, CTC-2 accumu-
lates during the reaction and at equilibrium, whereas
CTC-1 evolves rapidly into other FNR states. Forma-
tion of these CTCs appears necessary for efficient
hydride transfer and the relative conformation and ori-
entation of FNR and NADP
+
⁄ H during the interac-
tion are critical [121]. Hydride transfer in systems
involving flavins and pyridine nucleotides is highly
dependent on the approach and colinear orientation
of the N5 of the flavin, the hydride to be transferred,
and C4 of the nicotinamide. In FNR, displacement
of the C-terminal Tyr appears to be required for the
interaction to occur [117,118]. The data reported to

sq
are perturbed by NADP
+
[55,72], lower nicotinamide
occupancy of the active site is expected in AnFNR
relative to higher plant FNRs. Therefore, differences
in nicotinamide binding to the active sites cannot be
discounted.
FNR catalytic site
The structure of the catalytically competent
FNR:NADP
+
conformation indicates that in AnFNR,
S80, C261, E301 and Y303 constitute the FNR cata-
lytic site [59,117]. Y303 plays distinct and complemen-
tary roles during the catalytic cycle by lowering the
affinity for NADP
+
⁄ H to levels compatible with turn-
over, by stabilizing the flavin semiquinone required for
electron splitting and by modulating the electron trans-
fer rates [59,117,118]. Moreover, a role in providing
adequate orientation between the reacting rings might
be envisaged [128]. S80 and C261 contribute to the
efficient flavin:nicotinamide interaction through the
production of CTCs during hydride transfer (in
spFNR S96 and C272) [96,129]. This Ser also contrib-
utes to semiquinone stabilization, and the volume of
the Cys residue modulates the enzyme catalytic effi-
ciency [119]. E301 has been studied in AnFNR and

which substrates are added is not important, although
Fd and Fld lower the affinity for NADP
+
and occupa-
tion of the NADP
+
-binding site weakens the Fd:FNR
M. Medina Flavoproteins in photosynthetic electron transfer
FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3949
and Fld:FNR complexes [70,132]. The two binding
sites are not completely independent, and the overall
reaction is proposed to work in an ordered two-sub-
strate process, with the pyridine nucleotide binding
first [70,133]. Complex formation between Fd
rd
and
FNR:NADP
+
was found to increase the rate of elec-
tron transfer by facilitating the rate-limiting step of the
process – dissociation of the product (Fd
ox
) [127].
Thus, in the system involving Fd, negative cooperativi-
ty in the ternary interaction is translated into positive
cooperativity at the kinetic level. However, some key
features of the process remain to be explained in
molecular terms because the expected molecular move-
ments are not apparent and, although reversibile, dif-
ferent mechanisms seem to apply in each direction

gene helps to build multidomain proteins [134,138]. A
photosynthetic function was first proposed for FNR,
but flavoproteins with FNR activity have been
described in chloroplasts, phototropic and heterotro-
phic bacteria, apicoplasts, and animal and yeast mito-
chondria [139]. Two unrelated families of proteins can
be found in these enzymes: the plant type and the glu-
tathione reductase type [126]. Based on their structural
and functional properties, plant-type FNRs are classi-
fied as plastidic type and bacterial type. Plastidic
FNRs efficiently catalyse electron transfer and hydride
transfer between low-potential one-electron carriers
and NADP
+
⁄ H, usually participating in the produc-
tion of NADPH. Bacterial FNRs generally exhibit
considerably slower turnover, provide the cell with
reduced electron carriers and are examples of novel
methods of FAD and NADP
+
⁄ H binding. However,
their structures, the particular residues involved in
FAD binding and the residues at the catalytic centre
are well conserved [91]. In addition, all plant-type
FNRs may share a similar catalytic mechanism [140].
The general fold found in FNR is also present in
other enzymes. Many of these enzymes are multidomain
proteins that, in addition to the FNR-like domain, also
contain Fd- or Fld-like domains. These proteins contain
FAD (or FMN) and a FMN or Fe–S protein, and shut-

-dependent
members are related to the different energies required
to produce stacking of the nicotinamide at the re-face
to FAD [119,138]. These observations are compatible
with a mechanism in which the initial interactions
between the enzyme and 2¢P-AMP must evolve
towards the production of alternative structures for
each protein. The fine-tuning of the enzyme catalytic
efficiency is governed by the distance between and
mutual orientation of the N5 of FAD and the nicotin-
amide C4. Therefore, it is reasonable to suppose that
ancestral FNR adapted its NAD(P)
+
⁄ H-binding site,
modulating unique orientations to adapt its efficiency
to the coenzyme oxidation or reduction rates required
in each particular electron transfer chain.
Applications of current knowledge
about Flds and FNRs
The NADPH-producing electron transfer chain has
been used to explore the possibility of redesigning
Flavoproteins in photosynthetic electron transfer M. Medina
3950 FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS
existing electron transfer systems so that they can per-
form functions other than that for which they were
synthesized [148,149]. Strategies to engineer stress tol-
erance in plants based on the typical stress response of
photosynthetic micro-organisms are underway [150–
152]. The change in the enzyme specificity with respect
to its coenzyme is another example of redesign. FNR

thank to my current and former PhD students, Dr M.
Martı
´
nez-Ju´ lvez, Dr J. Tejero, Dr I. Nogue
´
s, Dr S.
Frago, J. R. Peregrina, G. Gon
˜
i, A. Serrano, B. Her-
guedas and I. Lans for their collaboration and interest
to better understand the FNR system and for all they
teach me everyday.
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