MUTATION OF THE HEME-BINDING CREVICE OF FLAVOCYTOCHROME-B2 FROM SACCHAROMYCES-CEREVISIAE - ALTERED HEME POTENTIAL AND ABSENCE OF REDOX COOPERATIVITY BETWEEN HEME AND FMN CENTERS

被引:18
作者
KAY, CJ
LIPPAY, EW
机构
[1] Bioscience Division of General Atomics, 92186, San Diego, California
关键词
D O I
10.1021/bi00161a015
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
Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme. Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction [Tegoni, M, Janot J.-M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342], while laser flash photolysis indicated pyruvate was essential [Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555]. To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (E(m)) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded E(m) values (pH 7.0, 25-degrees-C) of +8 and -28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate. These results indicated the absence of redox cooperativity between FMN and heme. Turnover numbers with electron acceptors ferricyanide (311 and 304 s-1, WT and L36I) and cytochrome c (211 and 192 s-1, WT and L36I) were similar, indicating lack of rate limitation by FSQ to heme electron transfer. Pyruvate inhibition with cytochrome c as acceptor was competitive in WT and L36I forms and was inconsistent with inhibition by pyruvate ligation of FSQ.
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页码:11376 / 11382
页数:7
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