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Titolo:
AMINO-ACID-RESIDUES IN ANABAENA FERREDOXIN CRUCIAL TO INTERACTION WITH FERREDOXIN NADP- SITE-DIRECTED MUTAGENESIS AND LASER FLASH-PHOTOLYSIS( REDUCTASE )
Autore:
HURLEY JK; SALAMON Z; MEYER TE; FITCH JC; CUSANOVICH MA; MARKLEY JL; CHENG H; XIA B; CHAE YK; MEDINA M; GOMEZMORENO C; TOLLIN G;
Indirizzi:
UNIV ARIZONA,DEPT BIOCHEM TUCSON AZ 85721 UNIV ARIZONA,DEPT BIOCHEM TUCSON AZ 85721 UNIV WISCONSIN,DEPT BIOCHEM MADISON WI 53706 UNIV ZARAGOZA,DEPT BIOCHEM & MOLEC & CELLULAR BIOL ZARAGOZA SPAIN
Titolo Testata:
Biochemistry
fascicolo: 36, volume: 32, anno: 1993,
pagine: 9346 - 9354
SICI:
0006-2960(1993)32:36<9346:AIAFCT>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
INTRACOMPLEX ELECTRON-TRANSFER; IONIC-STRENGTH DEPENDENCE; CYTOCHROME-C PEROXIDASE; SP STRAIN PCC-7120; ESCHERICHIA-COLI; CHEMICAL MODIFICATION; TRANSIENT KINETICS; FLAVODOXIN; COMPLEXES; BINDING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
54
Recensione:
Indirizzi per estratti:
Citazione:
J.K. Hurley et al., "AMINO-ACID-RESIDUES IN ANABAENA FERREDOXIN CRUCIAL TO INTERACTION WITH FERREDOXIN NADP- SITE-DIRECTED MUTAGENESIS AND LASER FLASH-PHOTOLYSIS( REDUCTASE )", Biochemistry, 32(36), 1993, pp. 9346-9354

Abstract

Ferredoxin (Fd) functions in photosynthesis to transfer electrons from photosystem I to ferredoxin-NADP+ reductase (FNR). We have made several site-directed mutants of Anabaena 7120 Fd and have used laser flash photolysis to investigate the effects of these mutations on the kinetics of reduction of oxidized Fd by deazariboflavin semiquinone (dRfH.) and the reduction of oxidized Anabaena FNR by reduced Fd. None of the mutations influenced the second-order rate constant for dRfH. reduction by more than a factor of 2, suggesting that the ability of the [2Fe-2S] cluster to participate in electron transfer was not seriously affected. In contrast, a surface charge reversal mutation, E94K, resulted in a 20 000-fold decrease in the second-order rate constant for electron transfer from Fd to FNR, whereas a similar mutation at an adjacent site, E95K, produced little or no change in reaction rate constant compared to wild-type Fd. Such a dramatic difference between contiguoussurface mutations suggests a very precise surface complementarity at the protein-protein interface. Mutations introduced at F65 (F651 and F65A) also decreased the rate constant for the Fd/FNR electron transferreaction by more than 3 orders of magnitude. Spectroscopic and thermodynamic measurements with both the E94 and F65 mutants indicated that the kinetic differences cannot be ascribed to changes in gross conformation, redox potential, or FNR binding constant but rather reflect theprotein-protein interactions that control electron transfer. Several mutations at other sites in the vicinity of E94 and F65 (R42, T48, D68, and D69) resulted in little or no perturbation of the Fd/FNR interaction. Kinetic experiments with the heterocyst Fd from Anabaena 7120, which functions in nitrogen fixation, are consistent with the above mutagenesis results and also indicate that Y98, which is also closely adjacent to E94 and F65, is not a critical residue for electron transfer to FNR. These results provide clear evidence for a high degree of localization and specificity in the interface region between the two proteins which is involved in the electron transfer process.

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Documento generato il 04/07/20 alle ore 17:36:40