Biomedical subjects

C Gómez-Moreno

Publications and source records attributed to C Gómez-Moreno.

At least 19 recordsLinked to original sources

PubMed2006

Point mutations in protein globular domains: contributions from function, stability and misfolding.

Several contrasting hypotheses have been formulated about the influence of functional and conformational properties, like stability and avoidance of misfolding, on the evolution of protein globular domains. Selection at functional sites has been suggested to be detrimental to stability or coupled to it. Avoidance of misfolding may be achieved by discarding misfolding-prone sequences or by maintaining a stable native state and thus destabilizing partially or fully unfolded states from which misfolding can take place. We have performed a hierarchical analysis of a large database of point mutations to dissect the relative contributions of function, stability and misfolding in the evolution of natural sequences. We show that at catalytic sites, selection for function overrules selection for stability but find no evidence for an anticorrelation between function and stability. Selection for stability plays a secondary role at binding sites, but is not fully coupled to selection for function. Remarkably, we did not find a selective pressure against misfolding-prone sequences in globular proteins at the level of individual positions. We suggest that such a selection would compromise native-state stability due to a correlation between the stabilities of native and misfolded states. Stabilization of the native state is the most frequent way in which natural proteins avoid misfolding.

Amyloid↗

PubMed2004

Analysis of the interaction of a hybrid system consisting of bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119.

The mitochondrial steroid-hydroxylating system in vertebrates and the NADPH producing electron transfer chain in photosynthetic organisms contain structurally and functionally similar components. Examination of a potential hybrid reconstitution of the electron transfer chain between different components of both systems could help to improve our knowledge on protein-protein interaction and subsequent electron transfer. Here we analyzed the interaction between bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119. Optical biosensor as well as steady state and fast kinetic experiments showed their ability to form distinct productive complexes. Compared with the corresponding physiological systems the electron transfer is rather slow, probably due to the lack of specificity at the interaction surface.

Animals↗

PubMed2001

Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal complex formation and electron transfer to ferredoxin.

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.

Amino Acid Sequence↗

PubMed2001

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria.

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.

Anabaena↗

PubMed2000

Highly nonproductive complexes with Anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin:NADP+ reductase.

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena function in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is extremely impaired relative to the reaction with wt FNR, and the dependency of k(obs) on E139K concentration shows strong upward curvature at protein concentrations > or = 10 microM. However, at values of mu > or = 200 mM, reaction rates approach those of wild-type FNR, and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR concentrations and low mu values, the dependency of k(obs) on E139Q concentration shows a smaller degree of upward curvature at mu = 12 and 100 mM and shows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 microM at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are explained by a mechanism in which the mutual orientation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increased E139K concentrations, the added mutant FNR reacts via a collisional interaction with the reduced Fd present in the unreactive complex. The et reactivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a negative charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.

Amino Acid Substitution↗

PubMed2000

Structural basis of the catalytic role of Glu301 in Anabaena PCC 7119 ferredoxin-NADP+ reductase revealed by x-ray crystallography.

The three-dimensional crystal structure of the Glu301Ala site-directed mutant of ferredoxin-NADP+ reductase from Anabaena PCC 7119 has been determined at 1.8A resolution by x-ray diffraction. The overall folding of the Glu301Ala FNR mutant shows no significant differences with respect to that of the wild-type enzyme. However, interesting conformational changes are detected in the side chain of another glutamate residue, Glu139, which now points towards the FAD cofactor in the active center cavity. The new conformation of the Glu139 side chain is stabilized by a network of five hydrogen bonds to several water molecules, which seem to hold the carboxylate side chain in a rather fixed position. This interacting network connects the Glu139 side chain to the Ser80 side chain through a series of three water molecules. These observations are discussed in terms of the reactivity of Glu301Ala ferredoxin-NADP+ reductase towards its substrates, and the role of Glu301 in the catalysis is re-examined. Moreover, a structural explanation of the different reoxidation properties of this mutant is given on the basis of the reported structure by modeling the hypothetical flavin C(4a)-hydroperoxide intermediate. The model shows that the distal oxygen of the peroxide anion could be in an appropriate situation to act as the proton donor in the reoxidation process.

Anabaena↗

PubMed2000

A redox-dependent interaction between two electron-transfer partners involved in photosynthesis.

Ferredoxin:NADP+:reductase (FNR) catalyzes one terminal step of the conversion of light energy into chemical energy during photosynthesis. FNR uses two high energy electrons photoproduced by photosystem I (PSI) and conveyed, one by one, by a ferredoxin (Fd), to reduce NADP+ to NADPH. The reducing power of NADPH is finally involved in carbon assimilation. The interaction between oxidized FNR and Fd was studied by crystallography at 2.4 A resolution leading to a three-dimensional picture of an Fd-FNR biologically relevant complex. This complex suggests that FNR and Fd specifically interact prior to each electron transfer and disassemble upon a redox-linked conformational change of the Fd.

Anabaena↗

PubMed1999

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.

Amino Acid Substitution↗

PubMed1999

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NADP+ reductase: site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein-protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.

Anabaena↗

PubMed1999

Ferredoxin-NADP(+) reductase uses the same site for the interaction with ferredoxin and flavodoxin.

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.

Anabaena↗

PubMed1998

Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer.

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The results reported herein reveal important conceptual information about the interaction of FNR with its substrates. A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.

Alanine↗

PubMed1998

Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer.

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.

Amino Acid Sequence↗

PubMed1998

Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119.

The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.

Anabaena↗

PubMed1998

Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis.

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.

Amino Acids↗

PubMed1998

Protein-protein interaction in electron transfer reactions: the ferredoxin/flavodoxin/ferredoxin:NADP+ reductase system from Anabaena.

Electron transfer reactions involving protein-protein interactions require the formation of a transient complex which brings together the two redox centres exchanging electrons. This is the case for the flavoprotein ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena, an enzyme which interacts with ferredoxin in the photosynthetic pathway to receive the electrons required for NADP+ reduction. The reductase shows a concave cavity in its structure into which small proteins such as ferredoxin can fit. Flavodoxin, an FMN-containing protein that is synthesised in cyanobacteria under iron-deficient conditions, plays the same role as ferredoxin in its interaction with FNR in spite of its different structure, size and redox cofactor. There are a number of negatively charged amino acid residues on the surface of ferredoxin and flavodoxin that play a role in the electron transfer reaction with the reductase. Thus far, in only one case has charge replacement of one of the acidic residues produced an increase in the rate of electron transfer, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 of Anabaena ferredoxin results in virtually the complete loss of ability to transfer electrons. Charge-reversal of positively charged amino acid residues in the reductase also produces strong effects on the rate of electron transfer. Several degrees of impairment have been observed, the most significant involving a positively charged Lys at position 75 which appears to be essential for the stability of the complex between the reductase and ferredoxin. The results presented in this paper provide a clear demonstration of the importance of electrostatic interactions on the stability of the transient complex formed during electron transfer by the proteins presently under study.

Amino Acids↗

PubMed1997

One- and two-dimensional ESEEM spectroscopy of flavoproteins.

One- and two-dimensional (1D and 2D) electron spin echo envelope modulation (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anionic semiquinone state of cholesterol oxidase from Brevibacterium sterolicum. High-resolution crystal structures are available for all these proteins. Three- and 4-pulse ESEEM and hyperfine sublevel correlation spectroscopy (HYSCORE) techniques at X-band were used. HYSCORE spectra showed correlations between transitions caused by interaction of the isoalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei. Measurements of isotopic labeled samples ([15N]FMN flavodoxin and [2H]flavodoxin) allowed the assignment of all the detected transitions to nuclei belonging to the FMN cofactor group. Interactions of nitrogens in positions 1 and 3 of the isoalloxazine ring were determined to have isotropic hyperfine coupling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied. Information about the quadrupolar term of these nuclei was also obtained. An intense correlation in the negative quadrant was detected. It has been associated to the strongly interacting N(10) nucleus. The complete hyperfine term parameters (including the sign) were obtained from detailed analysis of this signal, being the quadrupolar parameter, K, also estimated. Another correlation in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected. Its interaction parameters were also determined. This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particular utility for detecting and assigning nuclear transition frequencies in flavoprotein semiquinones. Moreover, the results reported here are complementary to ENDOR studies, and both techniques together provide an important tool for obtaining information about spin distribution in the flavin ring of flavoproteins in the semiquinone state.

Anabaena↗

PubMed1997

Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials.

Flavodoxins are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule as the redox-active center. The redox potentials of the flavin nucleotide are profoundly altered upon interaction with the protein. In Anabaena flavodoxin, as in many flavodoxins, the flavin is sandwiched between two aromatic residues (Trp57 and Tyr94) thought to be implicated in the alteration of the redox potentials. We have individually replaced these two residues by each of the other aromatic residues, by alanine and by leucine. For each mutant, we have determined the redox potentials and the binding energies of the oxidized FMN--apoflavodoxin complexes. From these data, the binding energies of the semireduced and reduced complexes have been calculated. Comparison of the binding energies of wild-type and mutant flavodoxins at the three redox states suggests that the interaction between Tyr94 and FMN stabilizes the apoflavodoxin--FMN complex in all redox states. The oxidized and semireduced complexes are, however, more strongly stabilized than the reduced complex, making the semiquinone/hydroquinone midpoint potential more negative in flavodoxin than in unbound FMN. Trp57 also stabilizes all redox forms of FMN, thus cooperating with Tyr94 in strong FMN binding. On the other hand, Trp57 seems to slightly destabilize the semireduced complex relative to the oxidized one. Finally, we have observed that reduction of mutants lacking Trp57 is slow relative to that of wild-type or mutants lacking Tyr94, which suggests that Trp57 could play a role in the kinetics of flavodoxin redox reactions.

Anabaena↗

PubMed1997

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants.

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

Anabaena↗