Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase

Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (∼1%), whereas unexpectedly the E312A reductase was 10–100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form. Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (∼1%), whereas unexpectedly the E312A reductase was 10–100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form. ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) oxidized FNR reduced FNR neutral FAD semiquinone ferredoxin I Michaelis complex charge-transfer complex 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride 2′-monophosphoadenosine-5′-diphosphoribose 3-acetylpyridine adenine dinucleotide phosphate 3-aminopyridine adenine dinucleotide phosphate thionicotinamide adenine dinucleotide phosphate 5-carba-5-deazariboflavin. Ferredoxin-NADP+ reductase (FNR)1 from plants and cyanobacteria fulfils the role of electrical switch between one- and two-electron transfer processes during NADP+ photoreduction in the photosynthetic electron transport chain (1Zanetti G. Aliverti A. Muller F. Chemistry and Biochemistry of Flavoenzymes. 2. CRC Press, Boca Raton, FL1991: 305-315Google Scholar, 2Arakaki A.K. Ceccarelli E.A. Carrillo N. FASEB J. 1997; 11: 133-140Crossref PubMed Scopus (138) Google Scholar). FNR became the structural prototype of a large family of structurally related flavoenzymes since the resolution of its three-dimensional structure, which highlighted a novel flavin binding fold (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar). Indeed, FNR-like modules are building blocks for constructing both simple and complex flavoproteins with the most varied biological functions not only in prokaryotes and plants but also in animals (4Correll C.C. Ludwig M.L. Bruns C.M. Karplus P.A. Protein Sci. 1993; 2: 2112-2133Crossref PubMed Scopus (159) Google Scholar, 5Karplus P.A. Bruns C.M. J. Bioenerg. Biomembr. 1994; 26: 89-99Crossref PubMed Scopus (99) Google Scholar). Generally, the members of the family are highly specific either for NAD+or NADP+, whereas they are more permissive with respect to the electron acceptor. The non-physiological reactions catalyzed in vitro by FNR can be divided into two half-reactions corresponding to transfer of a hydride between NADPH and FAD, and transfer of single electrons between reduced FAD and electron carriers (A), such as Fd (cytochromec), ferricyanide, and presumably INT, according to Scheme 1. FNRox+NADPH→FNRred+NADP+(reductive half­reaction)FNRred+nAox→FNRox+nAred(oxidative half­reaction)Scheme 1 The stoichiometric coefficient n can be 1 or 2, depending on the type of electron acceptor. The reductive half-reaction of FNR and other FNR family members occurs in discrete steps, which involve two Michaelis complexes (MC) and two charge-transfer complexes (CT) (6Batie C.J. Kamin H. J. Biol. Chem. 1984; 259: 11976-11985Abstract Full Text PDF PubMed Google Scholar, 7Batie C.J. Kamin H. J. Biol. Chem. 1986; 261: 11214-11223Abstract Full Text PDF PubMed Google Scholar, 8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar, 9Gassner G.T. Wang L. Batie C. Ballou D.P. Biochemistry. 1994; 33: 12184-12193Crossref PubMed Scopus (48) Google Scholar, 10Gassner G.T. Ballou D.P. Biochemistry. 1995; 34: 13460-13471Crossref PubMed Scopus (30) Google Scholar), according to Scheme 2. FNRox+NADPH⇌FNRox­NADPHMC1-⇌[FNRox­NADPH]*-CT1⇌[FNRred­NADP+]*-CT2⇌-FNRred­NADP+-MC2⇌FNRred+NADP+Scheme 2 In order to clarify the mechanism of action of hydride transfer mediated by the enzyme through flavin-nicotinamide rings interaction, for which there was no support from crystal structures (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar, 11Bruns C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar, 12Serre L. Vellieux F.M.D. Medina M. Gómez-Moreno C. Fontecilla-Camps J.C. Frey M. J. Mol. Biol. 1996; 263: 20-39Crossref PubMed Scopus (131) Google Scholar), our group and others have carried out site-directed mutagenesis of the five conserved residues surrounding the isoalloxazine ring in the active center of FNR: Tyr-95, Ser-96, Cys-272, Glu-312, and Tyr-314 (spinach numbering). It was shown by changing Cys-272 to Ser that this residue, conserved in all the members of the family, is required for productive interaction of the nicotinamide ring of NADP+ with the flavin to facilitate hydride transfer between C-4 of nicotinamide and N-5 of the isoalloxazine (13Aliverti A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar). Replacement of Ser-96 with Gly or Val clearly interfered with the proper binding of the nicotinamide and with the stabilization of the transition state during hydride transfer between NADPH and FAD (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar). Substitution of Phe for Tyr-95, which interacts with the si face of the isoalloxazine and makes a H-bond to the ribityl 4′-OH group, did not alter significantly the physico-chemical properties of the enzyme (14Aliverti A. Pandini V.E. Sternieri F.A. Corrado M.E. Karplus P.A. Zanetti G. Mathis P. Photosynthesis: From Light to Biosphere. II. Kluwer Academic Publisher, Dordrecht, Netherlands1995: 653-656Google Scholar). 2A. Aliverti, Z. Deng, P. A. Karplus, and G. Zanetti, unpublished results. 2A. Aliverti, Z. Deng, P. A. Karplus, and G. Zanetti, unpublished results. Mutation of the chain the face of the was for it be replaced by or Phe with only a in that it did not have a role in Carrillo N. Ceccarelli E.A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). In this the role of Glu-312, a residue that to Ser-96 and been to a role as proton donor during the one-electron reduction of FNR by ferredoxin C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar). residue is conserved in all the members of the FNR family, reductase and reductase (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar). replaced Glu-312 with and but and Ala the chain and by kinetic of the mutants and the three-dimensional structures of of them lead to the that Glu-312 does not a role as the proton but is required for nicotinamide proper in the active have been G. Aliverti A. Curti B. Karplus P.A. and of Press, Scholar). INT, and NADP+ and from was from and from either or other of mutagenesis of the FNR was carried out according to the J.R. F. PubMed Scopus Google Scholar), the in vitro mutagenesis The was by the FNR in as A. Lübberstedt T. Zanetti G. Herrmann R.G. Curti B. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The of the for mutagenesis is in In to the in the Glu-312, to the this the carried a in the to a in order to facilitate The of the and the of by of the was according to the chain F. Sci. A. PubMed Scopus Google and the by of for are are in a The the FNR in the as for other FNR mutants (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar). and mutant from as (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar), with the only that on was as the of on was on The of the mutant was a with the or The coefficient of the flavin was by the FAD from the and the FAD, according to the in (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar). Protein and flavin on a FNR reduction was by one-electron from the semiquinone by as the electron P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was both in the and in of NADP+. carried out in on FNR in in the of and NADP+ was a of by and with mutant was to of to reduction of enzyme and of with a The be a of the redox of the enzyme FAD and as (13Aliverti A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar). with a with a the transfer between NADPH and FNR was by NADPH with oxidized in as (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar, A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar). The of the complexes of the and the mutant with oxidized Fd by the reductase with Fd in as in 8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google the of the complexes of the mutant with NADP+ and the with the FNR in to a of and of from the in the of to for as in 8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google FNR in to a of of and to as of FNR mutants and to for FNR (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar, 11Bruns C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar). These to group with and for a for a for the mutant to single on a PubMed Scopus Google with a as C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar). The of and was carried out for and Press, Scholar). The FNR structure 11Bruns C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Protein was as with residue from to the residue, and residue from Val to Phe T. H. J. Herrmann R.G. Scopus Google Scholar, 11Bruns C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar). electron also that residues surrounding as from as was in the mutant structure 8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google These corresponding between and the resolution and by by of the The and the are shown in II. the structure was C.M. Karplus P.A. J. Mol. Biol. 1995; 247: 125-145Crossref PubMed Scopus (171) Google Scholar), a was by this the a structure that was almost to the a between and and in are for the resolution in are for the resolution from in are for the resolution in a The mutant all in and to the for the enzyme The of the was by and by the of the in Indeed, only the as the of the enzyme, whereas the other a charge, The of the mutant was by from the mutants the absorption of the to be and for and E312L The flavin was in the mutants as in the but in the of the oxidized of the mutant with respect to the These are to by of Ser-96 with Val or Gly (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar), but of The E312L mutant is in that it the but not G. Aliverti A. Curti B. Karplus P.A. and of Press, Scholar). was with all but only of the Leu, or mutants no of the structures only are the active Generally, the of is in mutant structure (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar). with the structure, the isoalloxazine ring of FAD in residue with its The group of Ser-96, which to Glu-312 in the structure, from In the ring of FAD from residue with the The group of Ser-96 from the isoalloxazine also in the that the between the of Ser-96 and the chain and of from FAD, with the In the of the isoalloxazine ring is The chain of from Ser-96 by as as are to between the and of as either the The group of Ser-96 and it is the to of In addition, the between the and in all mutants by with the of the mutants for the either or as the electron the reductive half-reaction is in the whereas the one is with The kinetic the two electron In the of ferricyanide, all the mutants a reduced and for NADPH almost with respect to that of the enzyme, for the Ala this mutant was the most of the whereas the and the a and the mutant a for of the Ala mutant was also and is the as the all the the a of with from to of that of the The for and for NADPH in the of the Ala mutant is in that it a for that of of and mutant for the and reactions and for the and reactions and in the significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type for the and reactions and in the significantly from type significantly from type in a of the during enzyme reduction by NADPH was by in by two the corresponding to the of a charge-transfer complex between oxidized FAD and NADPH the corresponding to the by hydride of a charge-transfer complex between reduced FAD and NADP+ 8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google and A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scheme charge-transfer complexes the of the since was 2 a to that with with the that the was in the only one was corresponding to the with the reduction by NADPH of the other mutants can be as a with no rapid of 2, and all mutants the in the was with flavin reduction and is to the charge-transfer complex reduced of the of FAD reduction in the various it that the flavin redox is for the mutants in the order E312A The of flavin reduction for the various in are to the in the that the reductive half-reaction is impaired by the The of the reduction that the Ala mutant reduction is to of the other not The binding of NADP+ and its to FNR can be by in the absorption C.J. Kamin H. J. Biol. Chem. 1986; 261: 11214-11223Abstract Full Text PDF PubMed Google Scholar). studies not only but according to the of Aliverti (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar, A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar), the of a the to which the nicotinamide ring is in the active center of FNR by the chain of The NADP+, a of the nicotinamide and The of FNR is in A. to that of no in the active binding a that that by NADP+, but it was more mutants with NADP+, and the with the is more for mutants. NADP+ binding a to that by in the of and whereas the in the between the two of with the of the mutants is highlighted by of the by binding to the enzyme the with a to that by FNR with this whereas in the of the and mutants the is to by the other two with NADP+ and to of but of and the Glu-312 mutants are from FNR in interaction with NADP+, the are not for NADP+ in the of for all the for the which The for binding was only for the enzyme and was to be that for the photoreduction with the P. J. Biol. Chem. Full Text PDF PubMed Google in the and of of NADP+ been to in the redox of the flavin and to the of FAD semiquinone and charge-transfer complexes that can be by mutant The in the of NADP+ that for FNR the NADP+ is as is with the of electron all mutants the FAD more to the NADP+ redox only a in redox and the other mutants have of semiquinone and charge-transfer complex during photoreduction of and mutant both in the and of of of FAD semiquinone during enzyme photoreduction as a of FAD of charge-transfer during enzyme photoreduction as a of the in the of of FAD semiquinone during enzyme photoreduction as a of FAD of charge-transfer during enzyme photoreduction as a of the in the of in a to semiquinone in the of NADP+, only as as the in the other the of during photoreduction is by The only of NADP+ was to by the of in mutants the neutral semiquinone as does The of charge transfer and was by the during the The of of such complexes was in all the mutants for the the of was of that for FNR the of the of all of the mutants from that of FNR have shown that the FAD of FNR is it absorption the is a which absorption M.E. Aliverti A. Zanetti G. J. 1996; PubMed Scopus Google Scholar). the mutants and that the charge on the residue absorption more to that of the FNR a in the the of the flavin in mutants. no in the of the mutants for oxidized Fd not The functional of this interaction was by the reductase of which electron transfer from NADPH to Fd and to The in the of which is with the enzyme, was in the of the Ala and depending on the of of and G. Aliverti A. Curti B. Karplus P.A. and of Press, Scholar). In the of type and two mutants with two of with the of not with the of electron transfer to is and the mutants of the with from the which was only with the other the Ala mutant a to transfer electrons to the be that of have more to the isoalloxazine ring Glu-312 been to Ala or Gln, of the of a The of the mutant be it with to a of the reductive reductase of the and mutant in the and of significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type significantly from type in a The of our the of the mechanism of hydride transfer between flavin and in the active center of a prototype of a large family of flavoproteins (4Correll C.C. Ludwig M.L. Bruns C.M. Karplus P.A. Protein Sci. 1993; 2: 2112-2133Crossref PubMed Scopus (159) Google Scholar, 5Karplus P.A. Bruns C.M. J. Bioenerg. Biomembr. 1994; 26: 89-99Crossref PubMed Scopus (99) Google Scholar). The conserved active center residue of Glu-312, been to as a proton donor during the of and its role been by the functional and structural of mutants Glu-312 replaced by Asp, Gln, or The three-dimensional structures of and that of residue Glu-312 to in the active It is the mutant did not but its and properties that it is also not highly perturbed. The structural that the properties of the mutants can be in of of the The that Glu-312 is as the proton to FAD during FNR that the half-reaction be Scheme and of the large a between a chain and one that the of the chain with in a in of of the proton transfer the of the mutants both with and In the of the enzyme, the of the is the reductive whereas the of the is the one (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar, A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar). The of the mutants with the of FAD reduction by that the half-reaction of flavin reduction is for the of the mutants. The reductase of the mutants are that the half-reaction is not impaired by the of to a of the interaction between NADP(H) and flavin in the FNR active center to Glu-312 a between of the mutants and charge-transfer complex both in rapid studies and photoreduction The which is the most impaired no of charge transfer complex was also for the highly impaired mutant (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar). there is a of of FNR as in the of the other mutants (13Aliverti A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google Scholar), (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar), and the of or can be during photoreduction and enzyme reduction with NADPH by the of Glu-312 with Asp, which is to be a was more to the enzyme of most of the residue chain to Ala that which can be by are more simple which be conserved in the The most impaired mutant was the which and does not for to the for impaired nicotinamide binding in mutants from the with NADP+ and all to the mutants with it be that structural studies have shown that the of is by the of the and does not the nicotinamide interacts with the (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar). Aliverti (8Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (62) Google Scholar, A. Piubelli L. Zanetti G. Lübberstedt T. Herrmann R.G. Curti B. Biochemistry. 1993; 32: 6374-6380Crossref PubMed Scopus (57) Google have shown that the can be to the of proper nicotinamide in the in this is that more NADP+, to a of the thionicotinamide this NADP+ can be as a more probe of nicotinamide to more between the mutants with and activity, support that the by NADP+ binding in the of FNR is of the nicotinamide in the active in the of the of the chain charge is for the of the nicotinamide ring but is not for The that Glu-312 be required for proper binding of the nicotinamide ring and that most a binding mechanism is as for binding to M. Biochemistry. 1997; PubMed Scopus Google Scholar), can be It to be shown by x-ray of the complex this residue interacts with the nicotinamide group, as been in other J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1994; 2: Full Text Full Text PDF PubMed Scopus (99) Google Scholar, Full Text Full Text PDF PubMed Google Scholar). In to this on interaction, in the properties of the a complex role for the Glu-312 It that the FAD semiquinone is in all the mutants for the that the charge of the chain a of the of charge on flavin properties are the of the of the which that the of of is in the neutral mutants. of Glu-312 which makes it for of the reduced isoalloxazine to a mechanism by which the redox potentials be In a the photoreduction in the of NADP+ that the two-electron of the flavin more in all but the The that can be from to be on two The of for the enzyme is in with that by M.E. Aliverti A. Zanetti G. J. 1996; PubMed Scopus Google Scholar), and the of flavin reduction of the enzyme mutants as in the is in the the mutant a redox most FNR is but it be that studies are the redox in the of NADPH and it is the NADPH to of the mutants. In of the with the to Ala and not the of binding but the electron transfer are by a of It been that electron from or to Fd is through the FAD which is to (3Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (457) Google Scholar), and which is to the chain of The of the Ala and mutants that the is not in but involve a more specific of the Glu-312 Medina M. M. G. Gómez-Moreno C. Biochemistry. PubMed Scopus Google have on the properties of FNR the corresponding to Ala as as that mutant for and a rapid kinetic they carried out there was a in the of electron transfer from that the residue was residue for electron but not be specific its In our the Asp, Leu, and mutants have us to more specific on the of charge, size, and polarity of this on the the role of Glu-312 in Glu-312 does not role as a proton but it is in the proper binding of NADP(H). The of the E312A that is a for the chain in the with The charge of Glu-312 to the redox potentials of the flavin semiquinone to electron The Glu-312 chain a on Fd or on electron transfer to but a on the electron transfer between FNR and are to A. Vanoni for in the for the of