Lysine residues on ferredoxin‐NADP<sup>+</sup> reductase from <i>Anabaena</i> sp. PCC 7119 involved in substrate binding

Medina, Milagros; Méndez, Enrique; Gómez‐Moreno, Carlos

doi:10.1016/0014-5793(92)80014-8

Cited by 38 publications

(22 citation statements)

References 21 publications

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“…The surface of FPR displays a striking concentration of positive potential within and around a cavity bounded by the ribityl atoms of FAD, N10 of the flavin, N6 and N7 of adenine, the side chains of Arg 51 and Lys 258 , and the amides of Ala 52 and Val 256 (Fig. 12A).…”

Section: Interaction Of Fdi and Fpr With The Chemical Cross-linkingmentioning

confidence: 99%

“…Arg 51 is a conserved residue in the motif RxYS/T that is involved in FAD binding in all homologous oxidoreductase structures; however, AvFPR is unique in that a second basic residue that was identified as the cross-linking site, Lys 258 , also interacts with the FAD phosphates (27). The presence of Lys 258 causes Arg 51 to interact with only the FMN phosphate deeper within the cavity. The methyl groups of the dimethylbenzene portion of the flavin isoalloxazine ring and the side chain of Val 256 are adjacent to the cavity on the protein surface.…”

Section: Interaction Of Fdi and Fpr With The Chemical Cross-linkingmentioning

confidence: 99%

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Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Jung¹,

Roberts²,

Stout³

et al. 1999

Journal of Biological Chemistry

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In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPHferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys Azotobacter vinelandii ferredoxin I (AvFdI)1 is a small ironsulfur ([Fe-S]) protein that has been extensively characterized by x-ray crystallography and direct electrochemical and spectroscopic methods (1-6). This seven-iron ferredoxin contains two different types of [Fe-S] showing Ͼ90% identity to AvFdI (8 -13). Although it has been known for some time that FdI has a metabolic function unrelated to nitrogen fixation that is important for cell growth (14), the specific cellular function of FdI and related seven-iron ferredoxins in other organisms has yet to be determined.In 1988, it was reported that disruption of the fdxA gene that encodes FdI in A. vinelandii led to a dramatic increase in the levels of another small acidic protein (13). In order to investigate the mechanism of regulation by FdI, the small acidic protein that is overexpressed in response to fdxA deletion was purified and characterized (15), and the gene encoding the protein was cloned and sequenced (16). The protein was shown to be a M r ϳ29,000 NADPH-ferredoxin reductase that was designated FPR because its physical properties and amino acid sequence showed striking similarity to the FPR from Escherichia coli (15,17,18). In E. coli, the protein appears to be part of a system that activates at least three different enzymes involved in anaerobic metabolism: anaerobic ribonucleotide reductase (19), pyruvate formate-lyase (20), and cobalamin-dependent methionine synthase (21). These activation reactions not only require NADPH and FPR but also ferredoxin or flavodoxin and S-adenosylmethionine. In those systems, FPR functions by mediating electron transfer between NADPH and the ferredoxin or flavodoxin (22). In E. coli, the fpr gene is activated in response to oxidative stress by the SoxRS regulon (23). In A. vinelandii, the fpr gene is similarly activated in response to oxidative stress; however, in that case the system is very specific for AvFdI (24, 25), a ferredoxin type that is not found in E. coli.Here, we have examined the possibility that in A. vinelandii both the metabolic and regulatory functions of FdI involve direct and highly specific interactions between FdI and FPR. As confirmed by both chemical cross-linking and spectroscopic characterization, AvFdI forms a complex with AvFPR at the stoichiometric ratio of 1:1 and the ϩ cluster of AvFdI is specifically reduced by NADPH via FPR. Recently, the x-ray structures of both E. coli and A. vinelandii FPR have been determined (26, 27). Taken together, these data are used to mo...

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Section: Interaction Of Fdi and Fpr With The Chemical Cross-linkingmentioning

confidence: 99%

Section: Interaction Of Fdi and Fpr With The Chemical Cross-linkingmentioning

confidence: 99%

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Jung¹,

Roberts²,

Stout³

et al. 1999

Journal of Biological Chemistry

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“…We did not see protection of either of these residues. In the enzyme from Anabaena, Lys294, equivalent to Lys305 of spinach FNR, seems to be involved in Fd binding (Medina et al, 1992b).…”

Section: Why Did the Two Reagents Not Reveal Protection Of The Same Rmentioning

confidence: 99%

Ferredoxin binding site on ferredoxin: NADP⁺ reductase

Jelesarov

Pascalis

Koppenol

et al. 1993

European Journal of Biochemistry

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The chloroplast enzyme ferredoxin : NADP' reductase (FNR) catalyzes the reduction of NADP' by ferredoxin (Fd). FNR and Fd form a 1 : 1 complex that is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. To localize lysine residues at the Fd binding site of FNR, the FNR:Fd complex (both proteins from spinach) was studied by differential chemical modification. In a first set of experiments, free FNR and the FNR:Fd complex were reacted with the N-hydroxysuccinimidyl ester of biotin. Biotinylated peptides and non-biotinylated peptides were separated on monovalent avidin-Sepharose and purified by high-performance liquid chromatography. Two peptides containing Lysl8 and Lys153, respectively, were less biotinylated in complexed FNR than in free FNR. In a second set of experiments, free and complexed FNR were treated with 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) to obtain coloured lysine-modified FNR. Protection of Lysl53 was again found by modification with S-DABITC. In addition, Lys33 and Lys35 were less labelled in the S-DABITC-modified, Fd-bound enzyme. FNR modified in the presence, but not in the absence, of Fd was still able to bind Fd, indicating that the Fd-protected residues are involved in the formation of the Fd: FNR complex.The lysine residues disclosed by differential modification surround the positive end of the molecular dipole moment (558 Debye -1.85 X lopz7 Cm) and are located in a domain of strong positive potential on the surface of the FNR molecule. This domain we had proposed to belong to the binding site of FNR for Fd [De

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“…The third patch is very large, including residues Lys 348 , Lys 349 , Lys 352 , and Lys 353 , while the fourth patch is located around the positively charged residue Lys 323 . Sequence comparison with spinach and Anabaena FNR show that most of these positively charged residues have been implicated in ferredoxin binding in the mentioned biochemical modification studies (45)(46)(47)(48)(49)(50)(51)(52).…”

mentioning

confidence: 96%

“…Nevertheless, these models were obtained by manually placing the ironsulfur cluster of Fd to the proposed binding cleft of FNR near the exposed portion of the flavin to account for the results of the biochemical studies (45)(46)(47)(48)(49)(50)(51)(52). Here we present an FNR-Fd docking model generated by computational molecular modeling.…”

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confidence: 99%

Crystal Structure of Paprika Ferredoxin-NADP+Reductase

Dorowski

Hofmann

Steegborn

et al. 2001

Journal of Biological Chemistry

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cDNA of Capsicum annuum Yolo Wonder (paprika) has been prepared from total cellular RNA, and the complete gene encoding paprika ferredoxin-NADP ؉ reductase (pFNR) precursor was sequenced and cloned from this cDNA. Fusion to a T7 promoter allowed expression in Escherichia coli. Both native and recombinant pFNR were purified to homogeneity and crystallized. The crystal structure of pFNR has been solved by Patterson search techniques using the structure of spinach ferredoxin-NADP ؉ reductase as search model. The structure was refined at 2.5-Å resolution to a crystallographic Rfactor of 19.8% (R free ‫؍‬ 26.5%). The overall structure of pFNR is similar to other members of the ferredoxin-NADP ؉ reductase family, the major differences concern a long loop (residues 167-177) that forms part of the FAD binding site and some of the variable loops in surface regions. The different orientation of the FAD binding loop leads to a tighter interaction between pFNR and the adenine moiety of FAD. The physiological redox partners [2Fe-2S]-ferredoxin I and NADP ؉ were modeled into the native structure of pFNR. The complexes reveal a protein-protein interaction site that is consistent with existing biochemical data and imply possible orientations for the side chain of tyrosine 362, which has to be displaced by the nicotinamide moiety of NADP ؉ upon binding. A reasonable electron transfer pathway could be deduced from the modeled structures of the complexes.

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Lysine residues on ferredoxin‐NADP⁺ reductase from Anabaena sp. PCC 7119 involved in substrate binding

Cited by 38 publications

References 21 publications

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Ferredoxin binding site on ferredoxin: NADP⁺ reductase

Crystal Structure of Paprika Ferredoxin-NADP+Reductase

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Lysine residues on ferredoxin‐NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding

Cited by 38 publications

References 21 publications

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

Ferredoxin binding site on ferredoxin: NADP+ reductase

Crystal Structure of Paprika Ferredoxin-NADP+Reductase

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Lysine residues on ferredoxin‐NADP⁺ reductase from Anabaena sp. PCC 7119 involved in substrate binding

Ferredoxin binding site on ferredoxin: NADP⁺ reductase