THE ROLE OF CYSTEINE RESIDUES OF SPINACH FERREDOXIN-NADP+ REDUCTASE AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

被引:61
作者
ALIVERTI, A
PIUBELLI, L
ZANETTI, G
LUBBERSTEDT, T
HERRMANN, RG
CURTI, B
机构
[1] UNIV MILAN,DIPARTIMENTO FISIOL & BIOCHIM GEN,VIA CELORIA 26,I-20133 MILAN,ITALY
[2] UNIV MILAN,CTR INTERUNIV STUDIO MACROMOLEC INFORMAZ,I-20133 MILAN,ITALY
[3] UNIV MUNICH,INST BOT,W-8000 MUNICH 19,GERMANY
关键词
D O I
10.1021/bi00076a010
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 muM-1 s-1) due to a substantial decrease of k(cat). NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (K(d) = 37 muM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD.NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.
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页码:6374 / 6380
页数:7
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