electron transfer between. NADP(H) and electron carrier proteins such as ferredoxin and fiavodoxin. Isoforms of this flavoprotein are present in chioroplasts,.
Plant-type
ferredoxin-NADP
structural ADRIAN
framework K. ARAKAKI,
EDUARDO
reductases: and a multiplicity
of functions
A. CECCARELLI,
CARRILLO’
Molecular Biology Division, PROMUBIE, Facultad de Ciencias de Rosario, Suipacha 531, (2000) Rosario, Argentina Ferredoxin-NADP (oxido)reductase (EC 1.18.1.2, FNR) is an FAD-containing enzyme that catalyzes the reversible electron transfer between NADP(H) and electron carrier proteins such as ferredoxin and fiavodoxin. Isoforms of this
ABSTRACT
flavoprotein dna,
are present
and bacteria
variety of redox redoxin-NADP
in chioroplasts,
in which
metabolic reductases
nutochon-
they participate in a wide pathways. Although fer-
have
been
thoroughly
investigated and their properties reviewed on several occasions, considerable advances in the understanding of these llavoenzymes have occurred in the last few years, including the characterization of cDNA and genomic clones encoding FNR proteins from plants, algae, vertebrates, and bacteria, determination of the atomic structure of a plant FNR at high resolution, and the expression of functional reductases in microorganisms like Escherichia coli and Saccharomyces cerevisiae. The aim of this article is to summarize information gained through
these recent developments, including the phylogenetic relationships among ferredoxin reductases and the key structural features of the plant FNR family. Other aspects such as the catalytic mechanism of FNR and the molecular events underlying biogenesis, intracellular sorting, folding, and holoenzyme assembly of this important flavoenzyme are also discussed in some detail. Ferredoxin-NADP
reductases
display
several
outstanding
properties
that make them excellent model proteins to address broad biological questions.-Arakaki, A. K., Ceccarelli, E. A., Carrillo, N. Plant-type ferredoxinNADP reductases: a basal structural framework and a multiplicity of functions. FASEB J. 11, 133140 (1997) Key Wordt: phylogeneticrelationships. flavoprotein FAD assembly . striwlure-function relationships
biogenesis
IN 1996, IT HAD BEEN 40 YEARS since Avron and Jagendorf (1) first described the isolation from spinach chloroplasts of a “TPNH diaphorase,” namely, an FAD-containing enzyme capable of transferring electrons to and from NADP(H). The role of this flavoprotein in catalyzing the last step of the photosynthetic electron transport chain was
0892-6638/97/0011-01
33/$01 .50 © FASEB
a basal
AND NESTOR Bioqufmicas
y Farmac#{233}uticas, Universidad
Nacional
clearly established in the next few years, largely through the brilliant work of the late D. I. Anion and his co-workers (2). They named the enzyme fenedoxin-NADP (oxido)reductase (EC 1.18.1.2, hereafter abbreviated FNR)2 and demonstrated that its physiological function in chloroplasts was to mediate the reversible electron transfer between two molecules of the obligatory one-electron carrier ferredoxin (Fd) and a single molecule of NADP(H):
2 FdFe12
+
NADP + H
2 FdFe3
+ NADPH
(1)
Equation 1 describes the only FNR reaction whose physiological relevance is established on firm grounds, leading to the inclusion of this enzyme into the functional class of flavoproteins called dehydrogenases/electron transferases (3). Plant reductases are able to mediate the oxido-reduction of ferredoxins from diverse origins, harboring various types of iron-sulfur clusters, and also of flavodoxins (Fld) that contain FMN as prosthetic group (reviewed in ref 4). Flavodoxins are synthesized in bacteria either constitutively or as a functional replacement of Fd when the microorganism is grown under iron-deficient conditions. Besides Fd (Fld), several other electron acceptors may participate in the FNR-mediated oxidation of NADPH, according to the following equation: NADPH
+
H
+
ii
A,,,
-
NADP
+
Ii A
(2)
in which A,,, and Ared represent the oxidized and reduced forms of the electron acceptor, respectively, and the term n equals one or two, depending on whether the oxidant behaves as a one-(ferricyanide, cytochromes) or a twoelectron carrier (NAD, indophenols). Ferredoxin is strictly required for cytochrome c reduction in vitro and also facilitates electron transfer to molecular oxygen. These reactions can be described as consisting of two hemireactions: an initial reduction of Fd as depicted in Eq. 1, followed by its rapid nonenzymatic reoxidation by Correspondence:
Area
Biologla
Molecular,
PROMUBIE.
Facultad
de Ciencias Bioqulmicas y Farmac#{233}uticas, Universidad Nacional de Rosario, Suipacha 531. (2000) Rosario, Argentina. 2 Abbreviations: Fd, ferredoxin; FNR. ferredoxin-NADP (oxido)reductase; Fld, flavodoxin; FNR.,, FNR semiquinone; GR, glutathione reductase; GST, glutathione S-transferase; preFNR, FNR precursor.
133
els in nonphototrophic plant tissues, including fruits and roots (10). The nonphotosynthetic reductases displayed essentially the same kinetic properties as the leaf enzyme and contained antigenic determinants in common with their chloroplast counterparts (10, 11). The presence of FNR in heterotrophic plant tissues is consistent with the existence of a redox pathway (comprising NADPH, ferredoxin, and FNR) that provides reducing equivalents to Fddependent enzymes involved in nitrogen metabolism, most conspicuously, nitrite reductase and glutamate synthase (10, 11). In cyanobacterial heterocysts, a similar pathway supplies the reducing power needed for dinitrogen fixation via nitrogenase (8). The isolation of FNR clones from various plant species and tissues confirmed that the chioroplast and heterotrophic reductases are encoded by different transcription units, and provided the first insights into the cis-acting regulatory elements of both types of genes (12, 13). It appears, therefore, that plant cells depend on ferredoxin and FNR irrespective of the type of plastid they contain. The chloroplast flavoprotein (and the FNRs from cyanobacterial vegetative cells) are unique in the sense that, under physiological conditions, they drive the reaction depicted in Eq. 1 toward NADP reduction, whereas the enzymes present in nonphotosynthetic plastids follow the general trend of the reductase family: the generation of reduced ferredoxin for a plethora of metabolic pathways. Finally, it has become increasingly clear that, in addition to their specific functions in the different organisms and cell types, FNR proteins may play a critical role in the concerted cellular defense against oxidative injury. The reductase from E. coli, for instance, is specifically induced by superoxide radicals. Inactivation of this flavoenzyme leads to mutant bacteria that are highly sensitive to radical propagating compounds (14). Complementation of the mutant cells with a cloned plant FNR gene restored the oxidative tolerance to wild-type levels, indicating that the reductase behaves as a toxic radical scavenger in the bacterial host (15). A comparable role for this flavoprotein in eukaryotes remains to be demonstrated.
the final electron acceptors. A summary of the reactions catalyzed by FNR is given in Table 1. The large amount of information accumulated in four decades of research on FNR may create the impression that the study of this flavoenzyme is almost exhausted and deserves little attention. In this review, we will show that the situation is quite the opposite. A number of unexpected properties of ferredoxin reductases have come to light in the last few years with broad implications for biological problems of general interest such as the detoxification of active oxygen species, the intracellular routing of precursor proteins, and the mode and tempo of FAD incorporation to flavoproteins.
THE
MANY USES OF THE FERREDOXJNNADP(H) ELECTRON TRANSFER IN LIVING ORGANISMS
After the original identification of a chloroplast FNR, equivalent flavoproteins were isolated from a variety of tissues and organisms, both phototrophic and heterotrophic. In nonphotosynthetic bacteria and eukaryotes, the reaction described by Eq. 1 is driven toward Fd or Fld reduction, providing electrons for metabolisms as diverse as steroid hydroxylation in mammalian mitochondria (5), methane oxidation in methanotrophic bacteria (6), reductive activation of biosynthetic enzymes in Escherichia coli and other procaryotes (ref 7 and references therein), as well as hydrogen and nitrogen fixation in anaerobes (8). In chloroplasts and cyanobacteria, ferredoxin is reduced by a photochemical reaction at the level of photosystem I. FNR then mediates electron transfer from this reduced iron-sulfur protein to NADP, with formation of the NADPH necessary for CO2 fixation and other biosynthetic pathways (ref 4 and references therein). Cyanobacterial flavodoxins are able to functionally replace Fd during NADP photoreduction (9). In addition to this chloroplast NADP reductase, FNR isoforms have been systematically detected at various lev-
TABLE
1. Different
activities
associated
with ferredoxin-NADP
oxidoreductases’ Electron donor
Electron
acceptor
Activity
Fd
NADP
NADPH
Potassium ferricyanide, indophenols.
Ferredoxin-NADP reductase Diaphorase
Molecular activity (s) 500 100-500”
viologens,
NADPH
tetrazolium Fd-cytochrome
NADPH NADPH
NAD 02, Fd-02
Data obtained erences therein. ceptor employed.
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1997
salts Cytochrome c reductase Transhydrogenase Oxidase
c
50-100
5-10 0.5
from refs 36, 37, 43, 44, and 54. See also ref 4 and refMolecular
The
activities
varied
FASEB Journal
according
to the electron
ac-
ARAKAKI
ET AL.
PLANT FERREDOX1N-NADP ARE THE PROTOTYPE OF STRUCTURALLY DISTINCT
REDUCTASES A FLAVOPROTEIN
FAMILY
Plant reductases are hydrophilic proteins of about 35 kDa containing 1 mol of noncovalently bound FAD per monomer (2, 16). Amino acid sequences have been determined (or deduced from nucleotide sequence data) for the FNR proteins from higher plant leaves and roots, from three eukaiyotic algae, and three cyanobacteria (13, 17-28). These reductases all belong to the group we shall name “plant-type” or “chioroplast-type” FNRs. They show a degree of sequence identity that varies rather widely (between 43 and 97%), although homology extends along the whole sequence. Clusters of well-conserved residues are kept at the NADP and FAD binding regions, the buried cores contributing to FNR folding and the carboxylterminal region involved in FAD shielding (29). Cyanobacterial FNRs contain in addition a 10 kDa aminoterminal extension whose sequence is related to the phycobiliprotein linker peptides (21, 23). This extra domain does not appear to participate in enzyme activity, and almost certainly folds independently from the remaining two domains recognized in chloroplast-type reductases (see below). The 3-dimensional structure of spinach FNR has been described at 1.7 ‘A resolution, providing insightful data to complement and extend information from biochemical results and known amino acid sequences (16, 29, 30). The flavoprotein is made up of two structural domains, each containing approximately 150 amino acids (Fig. 1). The FAD group binds to the amino-terminal domain whereas the carboxyl-terminal region harbors the NADP site (16). Each domain is a compact structure that binds the bulk of its respective nucleotide, although both cofactors also interact with some residues of the other domain. The large shallow cleft between the two domains provides for Fd binding whereas a 200”A3 cavity, located close to the protein surface and distant from the active site, is presumably involved in membrane attachment (30). Plant-type FNR appears to be the prototype for a twodomain structure that occurs in many enzymes that need to transfer electrons from a nicotinamide nucleotide, one at a time, to one-electron acceptors. This large family of flavoprotein reductases and oxidases is defined by six clusters of highly conserved residues, three of them belonging to the FAD (FMN) domain, and the remaining to the NAD(P) region (29, 31; see also Fig. 1A).The ferredoxin reductases from E. coli and some related proteobacteria display sequence identities of less than 20% with their chloroplast and cyanobacterial counterparts, although the similarity scores are well above 40% due to conservative replacements (7). Indeed, these flavoenzymes contain the six consensus sequences defined for the FNR prototype (Fig. 1A), indicating that they meet the structural requirements of the reductase family.
FERREDOXlN-NADP
REDUCTASES
A Spinacia oleracea Pisuni Sativum Vicia faba N. crysta11inw Oryza saliva (leaf) Oryza saliva (root) Zea says (root) Cyanophora paradoxa Voivox carteri C. reinhardtji Spirulina Synechococcus p. Anabaena sp. Escherichia coli
1
2
93RLYS
1eGVCS
3
4 234
‘71GTGIAP
5 0YMCGL
6 312EVT v.
i V I
v.
I
T.
I AIG
.A
T.
v.
...
ML. .5
T. .H.
B
FAD domain
NADP
domain
Figure
1. A) Conserved sequences in plant-type ferredoxin-NADP reductases. The six peptide segments that define the FNR structural family (16, 31) are aligned, showing (in bold) the amino acid residues involved in FAD (segments 1, 2, and 6) or NADP binding (segments 3-5). Numerals on the residues indicate their relative positions in the spinach mature FNR sequence (314 amino acids; ref 18). B) Schematic representation of the Ca polypeptide backbone of plant-type FNR. The computer graphic was based on X-ray crystal diffraction data for the spinach
enzyme
(30).
The
positions
of the
six
consensus
sequences
of
the FNR family (A) are indicated in bold, and the FAD cofactor is displayed in grey lines. N and C represent the amino and carboxylterminal regions of the protein, respectively. The dotted line indicates the first 18 residues of FNR that are not visible in the electron density map (30).
A different group of FNRs, which includes those present in mitochondria and some bacterial species, are clearly unrelated in sequence to both plant and E. colireductases (5), and do not display the chloroplast-type FNR conserved sequences. They cluster with the glutathione reductase (GR) family, consistent with the presence of more typical nucleotide binding domains (5, 16). The plant-type and the GR-type FNR progenies probably represent two different and independent origins that followed a convergent evolution to yield proteins with essentially the same enzymatic properties. When the NADP and fiavin domains were separately compared among plant-type FNRs, higher similarities (8% in average) were obtained for the NADP domain, confirming and extending an earlier observation by Ritchie et al.
135
(26). However, the phylogenetic trees generated by the sequences of the two domains (and of the entire reductase) reproduce the same topology within the plant, algal, and cyanobacterial FNR groups (Fig. 2), suggesting that the two binding regions evolved together from a unique ancestral gene. Neither the bacterial nor the mitochondrial reductases could be included in the tree due to their sequence divergence with respect to plant-type FNR. The most striking feature of the FNR phylogeny is, however, the unexpected relationship between the algal and root FNR groups. Although the small number of sequences considered precludes a conclusive explanation for this clustering, it suggests the occurrence of an ancient duplication event of the FNR gene preceding the separation of the algal and plant groups, followed by differential loss of the leaf-type gene in the algal lineage.
C Th
\7
INTO
THE
CATALYTIC
Rapid reaction studies have demonstrated that the FNRcatalyzed electron transfer from Fd to NADP proceeds through the formation of a ternary complex, leading to the proposed catalytic cycle shown in Fig. 3 (32). As illustrated, the reaction pathway involves two successive oneelectron reductions of the isoalloxazine, followed by a hydride transfer stereospecific for the A side of the nicotinamide ring (33), to produce NADPH (32). Several steps in the cycle are supported by extensive kinetic studies (refs 32, 34 and references therein). Bind-
Oryza Sativa
Hcia,
Oriza sativa (root) - Zea mays (root)
Spinacia oleracea Mesembryanthemum
Volvox carteri
crystallinum
Synechococcus
sp.
Spirulina
Anabaena
sp. 0.2 subst./tite sp..
Figure
2. Phylogenetic relationships of plant-type ferredoxin-NADP reductases based on amino acid sequence comparisons. An unrooted tree was constructed by the neighbor-joining distance method. Evolutionary distances among 13 FNR protein sequences (5, 7, 13, 17-28) were computed by the Dayhoff PAMOO1 matrix (62) over 331 aligned positions. The transit peptide sequences of the chloroplastand cyanelle-targeted FNRs and the 10 kDa amino-terminal domain of cyanobacterial reductases were not included in the comparisons. The length of each branch is proportional to the calculated evolutionary distance; the scale is indicated at the bottom. Confidence limits for the inferences obtained were determined by the bootstrap procedure. Numerals on each branch indicate the number of times that the two groups it defines occurred in 100 bootstrapped samples. The phylogenetic analysis was carried out using the PHYLIP package. version 3.5 (63).
136
Vol. 11
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1997
2
FNR, oxidized Ferredoxin, reduced Ferredoxin, oxidized NADPH
_
3J
NADP
6electron
K
0 Figure proposed
INSIGHTS MECHANISM
o
c
FNR, reduc FNR, semiquinone
3. The catalytic by Batie
pathway of ferredoxin-NADP and Kamin (32).
reductase
as
ing of Fd to FNR was found to impair the reaction of the flavoprotein with NADP(H), whereas occupation of the NADP binding site by nucleotides greatly increased the electron transfer from Fd to FNR, relieving the inhibition exerted by bound Fd,,, (32). NADP(H) facilitates the dissociation of oxidized ferredoxin molecules (reactions 4 and 8 in Fig. 3), which are the rate-limiting steps of the overall reaction (32). Thus, substrate attachment must be ordered, with NADP first and then Fd (Fig. 3). Charge-transfer species between FNROX and NADPH (35) were not observed during enzyme turnover, so that their participation in catalysis remains uncertain (32). The NADPH-driven reduction of Fd is expected to occur through a reversal of the catalytic pathway of Fig. 3, but the experimental basis for such a conclusion is scant. Massey et al. (35) reported on the participation of the fiavin semiquinone during FNR-mediated NADPH oxidation by artificial electron acceptors. Similar results were obtained in static experiments in which the flavoprotein was titrated anaerobically with NADPH (2, 36). Note that the number of electrons involved in FNR reduction shifted from one to two as the pH was raised (36). The one-electron reduction of enzyme-bound FAD at pH 6-8 is difficult to interpret in terms of the hydride transfer mechanism proposed in Fig. 3 (see below). As already mentioned, Fd (or Fid) reduction is normally coupled in vitro to a reaction in which the protein substrates are rapidly reoxidized by cytochrome c (reviewed in ref 4). This cytochrome c reductase activity (Table 1) yields a series of parallel straight lines in double reciprocal plots of 1/v vs. 1/[NADPH] (37). This type of behav-
ior is usually
interpreted
in terms of a two-step
transfer
“ping-pong” mechanism, although Massey (quoted in ref 37) has shown that a kinetic mechanism similar to that depicted in Fig. 3 can still produce parallel lines, depending on the relative rates of ternary complex formation and intramolecular electron transfer. Taking into account the rates observed in a number of systems (34, 38), it is
The FASEB Journal
ARAKAKI
ET AL.
reasonable to assume that the Fd05-FNR05-NADPH complex is rapidly converted into FNRSqNADP and Fdre (reactions 7 to 5 in Fig. 3). This might explain why neither the ternary complex nor the fully reduced flavin is significantly accumulated during enzyme turnover under steady-state conditions. Further research will be required to elucidate these questions. Complex formation between FNR and its ligands (particularly the extensive interaction with Fd) is expected to play a crucial role in catalysis (29), and has therefore been studied with considerable detail. Experiments using plant and bacterial proteins led to the conclusion that electrostatic forces are critical for the establishment of an FNRFd complex competent in electron transfer (34). However, the roles played by these forces are different in the various systems. Electrostatic interactions between the plant proteins are dominant in the formation and stabilization of an initial collision complex that is already optimized for electron transfer, whereas for the cyanobacterial FNR and Fd these interactions only lead to an approximately correct orientation, which is then rearranged into an optimal complex through nonelectrostatic forces (34). Covalent stabilization of the FNR-Fd complex could be accomplished through chemical cross-linking with watersoluble carbodiimides (39), allowing a more convenient way to study what would otherwise be a transient kinetic intermediate. The chimeric protein thus formed was shown to be a valid model of the in vivo noncovalent complex by numerous criteria, including catalytic efficiency, and led to the initial identification of several amino acid residues involved in protein-protein interaction (39). Based on these results, the protein-substrate binding site of the reductase could be described as a hollow surface decorated with several positively charged side chains to direct and interact with carboxylate groups in the acidic protein partners (30). The role of specific amino acids in the establishment of the FNR-Fd complex has been further confirmed by site-directed mutagenesis and chemical modification studies (reviewed in ref 38). As indicated in the introductory statement, FNR is also able to bind and transfer electrons to and from flavodoxins from various origins. From the properties of both electrostatic and covalently linked complexes, it has been concluded that Fid binds to the reductase in a way similar to that of ferredoxin (9, 40). Pueyo et al. (9) have shown that the electrostatic complexes formed when FNR and Fld differ in their redox states are stronger than those occurring when the two proteins are fully oxidized or fully reduced. These results suggest that the catalytic pathway of Fig. 3 may also be valid for the NADP(H)-flavodoxin electron transfer, although accumulation of FAD semiquinone in FNR during turnover was very low (9). Association of FNR with either Fd or Fld was shown to cause a shift in the redox potentials of the individual proteins in the thermodynamic direction that facilitates electron transfer (9, 32). Further insights into the catalytic mechanism of FNR have been obtained from the crystal structures of the oxidized and reduced forms of the flavoenzyme (16, 33). Fig-
FERREDOXINNADP*
REDUCTASES
ure 4 shows a model for binding of the NADP nicotinamide moiety, which is assumed to be stacked coplanar to the flavin ring (31). Karplus et a!. (16) have postulated that the phenol side chain of the carboxyl-terminal tyrosine has to be displaced to accommodate the nicotinamide upon nucleotide binding. However, mutagenesis studies of this residue suggest that the tyrosine may play a more active role in catalysis (41). It is possible that the phenol group helps to fix the nicotinamide in its proper position during enzyme turnover (31). Two other well-conserved residues (corresponding to Ser and Cys272 in spinach FNR) are proposed to interact respectively with N-5 of the flavin and C-4 of the nicotinamide, the two atoms involved in hydride transfer (Fig. 4; 31). Mutations on Ser resulted in a dramatic decrease of the FNR catalytic efficiency (42), consistent with the fundamental role assigned to this residue, whereas replacement of Cys272 caused only a moderate decline in activity, suggesting that the sulfhydryl group plays a supporting, but not crucial, role in catalysis (43). In good agreement with these results, the reduced structure of spinach FNR shows that Ser moves toward atom N-5 of FAD, whereas the isoalloxazine ring remains planar (33). FNR reduction involves protonation of N-5, which is not exposed to solvent. Possible sources for this proton are a buried water molecule close to this atom or the hydroxyl group of Ser, which in turn could obtain a proton from the surface-exposed side chain of G1u312 (33). In good agreement with this hypothesis, substitution of the glutamate residue by leucine resulted in a mutant FNR displaying less than 1% of the enzymatic activity of the wild-type flavoprotein (44).
REGULATION FERREDOXIN-NADP
OF THE
CHLOROPLAST REDUCTASE
ACTIVITY
Inside chloroplasts, FNR binds to the stromal surface of the thylakoid membrane (45). Different modes of membrane association may regulate FNR activity in vivo, since changes in the ratio of loosely and tightly bound enzyme correlate with NADP photoreducing activity and affect FNR kinetic parameters (4). Several lines of evidence indicate that a 17.5 kDa thylakoid protein is the most likely candidate for the tight binding site at the membrane surface (45-47). On the basis of biochemical properties and intraplastidic distribution, it has been argued that the FNR binding protein may be identical to a 16.5 kDa peptide previously characterized as a luminal component of the oxygen evolving system (48), but the question is still controversial. Chloroplast FNR is activated in vivo via a light-driven conformational change of the membrane-bound enzyme. The effect of light is mediated by the transmembrane proton gradient, and results in a dramatic increase in the affinity of the reductase for its physiological substrates, Fd and NADP. Further details on the activation process can be found in a number of reviews (4, 49), although there is not yet a molecular explanation to account for activity modulation. The light-
137
Ser96 Cys 272
Figure rings
4.
Postulated
interaction
in ferredoxin-NADP
between
reductase.
the nicotinamide
Adapted
from
and fiavin
ref 29.
dependent activation of thylakoid-bound FNR has been also shown to occur in cyanobacteria (50). The reductase from Chiamydornonas reinhardtii undergoes lysyl methylation in vivo (51), but the physiological relevance of this post-translational modification is unknown.
THE PRECURSOR NADP REDUCTASE
TRANSPORTED
OF
INTO
FERREDOXINAND IS CHLOROPLASTS
LEAF BINDS
FAD
Like most plastid proteins, chioroplast FNR is nuclear encoded and synthesized on free cytosolic polysomes as a higher molecular mass precursor (preFNR), containing an amino-terminal extension of ‘-5 kDa (19). This extension is called the transit peptide and allows the precursor protein to be targeted to and translocated across the plastid envelope. During or shortly after import, the transit peptide is cleaved off by a stromal protease and the mature protein binds to the thylakoid membranes (19). The development of various systems for the expression of plant FNR precursor and mature proteins in microorganisms opened new opportunities to investigate the molecular nature of precursors and the requirements for plastid import (52, 53). Expression of a pea preFNR gene in E. coli resulted in the accumulation of a fully active mature-sized reductase, indicating that the transit peptide was cleaved off in the bacterial host (52). The use of protease-deficient strains largely prevented this processing, but the precursor was totally insoluble (52, 54). Further information could be gained by expressing preFNR in E. coli as a carboxyl-terminal fusion to Schistosoma japonicum glutathione S-transferase (GST). The presence of a folded protein at the amino terminus largely
138
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prevented both transit peptide processing and preFNR precipitation. The fusion protein accumulated in soluble form, and the FNR precursor could be cleaved off from GST and purified to homogeneity (55). Isolated preFNR was able to assemble the prosthetic group FAD, which requires the formation of a specific 3-dimensional motif at the flavin binding domain (29, 41, 56). Limited proteolysis resulted in rapid removal of the transit peptide sequence, with accumulation of a protease-resistant core similar to that of the mature reductase. These results suggest that the precursor holoprotein is packed to a certain extent with protease-sensitive sequences limited to the transit peptide and amino-terminal regions (55). The precursor holoprotein could be translocated into isolated chloroplasts and processed to mature size (55). Maximal import rates were obtained by inclusion of leaf extracts in the assay or by denaturing the precursor with urea. Determination of the import kinetic indicates that the rate-limiting step is translocation of the protein and not its binding to plastid envelope receptors. The stimulation induced by soluble factors present in the leaf extracts is probably related to precursor unfolding (57). It is likely that assembly of the preFNR holoprotein after synthesis may increase protein stability and solubility, helping the precursor to reach the organelle. However, preFNR should be unfolded to be transported across the membrane according to current views on protein translocation (58). The transit peptide may facilitate preFNR unfolding by preventing the strong structuring effect induced by FAD incorporation. In line with this hypothesis, the secretion of a recombinant FNR from yeast cells was shown to be strongly stimulated by introduction of the FNR transit peptide between the yeast-specific secretion signal and the mature protein (53). This leads to another important question related to preFNR import: the site of FAD attachment. No information is available about chloroplast FAD uptake from the cytosol, where the prosthetic group is synthesized (59), or regarding the incorporation of the flavin moiety to apoproteins in plastids. The availability of a defined import system with recombinant preFNR offers good opportunities to address these questions.
MOLECULAR SPONTANEITY
CHAPERONES ASSIST DURING FERREDOXIN-NADP
REDUCTASE
HOLOENZYME
ASSEMBLY
The notion that protein folding is not a spontaneous process, determined solely by the primary structure and thermodynamic constraints, has gained momentum in recent years. In all living organisms and in all subcellular compartments, different classes of molecular chaperones have been shown to interact with newly synthesized peptides and to mediate their proper folding and assembly (60). Chaperone proteins belonging to the hsp#{244}O (GroE) and hsp70 (DnaK) classes are remarkably conserved along evolutionary lineages. These chaperones form complexes
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with folding intermediates of the newly synthesized peptides and assist the acquisition of a native conformation, presumably by preventing the target protein from following nonproductive folding pathways (60). Expression of FNR in an E. coliconditional mutant in which the GroE folding capabilities were completely interrupted caused a total breakdown of the holoenzyme assembly pathway, with aggregation and precipitation of the apoprotein. The effect was reverted by coexpression of a cloned groESL operon (54), suggesting that the products of the host groE genes are essential for productive folding and assembly of the eukaryotic reductase in the bacterial cell. We have obtained preliminary results indicating that plant FNR also requires chaperones of the DnaKIDnaJ family to acquire a functional holoenzyme conformation in
E. coli. The role played by the prosthetic group FAD in FNR folding is not clear. The flavin moiety is bound outside of the antiparallel n-barrel that makes up the core of the FAD binding domain (16, 29). Exposition of the prosthetic group to solvent is limited, since the isoalloxazine ring is largely shielded by the side chain of the carboxyl-terminal tyrosine. This amino acid is conserved in all FNR proteins described so far (see cluster 6 in Fig. 1A), and aromatic residues have been found in equivalent positions in other members of the FNR structural family (16, 29, 30). The terminal tyrosine actually belongs to the NADP binding domain, but it is located at the end of an -helixI-strand region that comprises the final 19 amino acids and folds back into the FAD binding site. The phenol ring of the tyrosine and the isoalloxazine ring of FAD are largely coplanar in such a way as to maximize ic-orbital overlap (16, 31). Substitutions of the carboxyl-terminal tyrosine were shown to destabilize the native conformation of FNR mutants with respect to the wild type (56). The decrease in thermodynamic stability correlated with the impairment of catalytic competence, with aromaticity being the most important factor to the function of the tyrosine (41, 56). Taken together, the results suggest that the presence of a tyrosine (or other aromatic residue) at the carboxyl-terminal position of FNR is required for proper docking and stabilization of the FAD group in its binding cleft during holoenzyme assembly in vivo. FAD attachment is probably needed for the formation of the core in the chaperoneassisted folding of FNR, as already demonstrated for some mitochondrial flavoproteins (61). Further work will be necessary to determine whether the FAD binding region of the protein play any relevant role in the interaction between FNR folding intermediates and the chaperone systems of the cell.
mensional structure of a plant reductase described at high resolution, we can expect rapid progress to be made in structure-function studies of this flavoprotein. Overexpression in microorganisms combined with site-directed mutagenesis will be particularly helpful in understanding the role of specific amino acids in substrate and cofactor binding, catalysis, and structural stability. Plant ferredoxin reductases possess a number of outstanding characteristics: they are stable, occur in a number of different tissues and organisms, and can be readily overproduced in transgenic cells. Their expression in eukaryotes involves the regulatory crosscurrents established between the organellar and the nuclear-cytoplasmic genetic compartments, and they need somewhere to assemble their prosthetic group. We therefore regard the information gained as not relevant solely to the understanding of FNR function per se. In our view, FNR proteins display all the conditions of an excellent model system to investigate broader biological problems-most remarkably, the biogenesis and assembly of fiavoproteins, and the mechanisms underlying organelle uptake of cytoplasmic precursors.
Some of the research presented here was carried out at the Molecular Biology Division, PROMUBIE, Rosario, supported by grants from Fundaci#{243}n Antorchas, Argentina. E.A.C. and N.C. are staff members, and A.K.A. is a Fellow from the Consejo Nacional de Investigaciones Cientfficas y Tecnologicas (CONICET, Argentina).
REFERENCES 1.
AND
FUTURE
2. 3. 4.
5.
6.
7.
1595 8.
9.
reduction
11.
FERREDOXIN-NADP’
REDUCTASES
Schrautemeier. B.. and Bohrne, H. (1985) A distinct ferredoxin for nitrogen fixation isolated from heterocysts of the cyanobacterium Anabaena carlabilis. FEBS Lett. 184, 304-308 Pueyo, J. J., GOmez-Moreno, C., and Mayhew, S. C. (1991) Oxidation-
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Anahaeno
PROSPECTS
In the last few years, research on the structure, function, and biogenesis of fenedoxin-NADP reductases has progressed at a remarkable pace. With the sequences from several different FNR proteins determined and the 3-di-
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10.
CONCLUSIONS
Avron,
ferredoxin-NADP 12.
oxiiioreduciase
and ferredoxin
synthesis
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plastids during nitrate assimilation. Plaru J. 3, 463-467 Oelmliller, R.. Bolle, C., Tyagi. A. K.. Niekrawietz. N., Breit, S., and Herrmann. R. C. (1993) Characterization of the promoter from the singlecopy gene encoding ferredoxin-NADP oxidoreductase from spinach. Mol. Gen. Genet. 237, 261-272
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15.
16.
17.
lB.
19.
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39.
40.
1
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41.
of ferredoxin-NADP reductase: prototype for a structurally zyme family. Science 251, 60-66
42.
novel flavoen-
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Jakowitsch, J., Bayer, M. C., Maier, T. L., L#{252}ttke, A., Gehhart, U. B.. Brandiner, M., Hamilton, B., Neumann-Spallari, C., Michatowski, C. B., Bohnert. H -.1.. Schenk, H. E. A., and Laffelhardt,W. (1993) Sequence analysis of pre-ferredoxin-NADP reductase cDNA from Gyanophora paradoxa specifying a precursor for nucleus-encoded cyanelle polypeptide. Plant Mol. BioI. 21, 1023-1033 Fillat. M. F.. Fiores, E., and C#{243}mez-Moreno, C. (1993) Homology of the Nterminal domain of the pet!! gene product from Anabaena sp. PCC 7119 to the cpcD phycobilisome linker polypeptide. Plant Mol. Bid. 22. 725-729 Lax. A. R. (1994) Viriafaba ferredoxin-NADP oxidoreductase precursor mRNA complete cds. Gene Bank Accession 1114956 Kitayama. M., Kitayama. K., and Togasaki. H. K. (1994) A cDNA clone encoding a ferredoxin-NADP reductase from Chlamydomonas reinhardtii. Plant Ph1siol. 106. 1715-1716
M. C., Shiraishi, N., Vrba, J., and Campbell, of a maize root transcript expressed in the to nitrate: characterization of a cDNA with homology to oxidoreductase. Plant Mol. Biol. 26, 679-690
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