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.
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.
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酿酒酵母黄细胞色素 b2 的血红素结合缝隙突变：血红素电位改变，血红素和 FMN 中心之间缺乏氧化还原协同作用。
DOI：
10.1021/bi00161a015
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Lippay,EW
中科院分区：
文献类型：
--
作者：
Kay,CJ;Lippay,EW
1992年9月10日收到的摘要：酵母黄细胞色素bi（EC 1.1）的动力学和热力学性质。2.3)被产物丙酮酸修饰，丙酮酸结合到人工黄素的黄素半醌（FSQ）形式上，降低了电子从FSQ转移到血红素的电子驱动力。丙酮酸盐抑制黄细胞色素bi，但丙酮酸盐连接的FSQ在分子内电子转移至血红素中的催化能力尚不清楚;一项动力学研究表明丙酮酸盐阻止该反应[Tegoni，M，Janot J. M.，& Labeyrie，F.（1990）Eur. J. Biochem. 190，329-342]，而激光闪光光解表明丙酮酸盐是必需的[步行者，M. C.的方法，& Tollin，G.（1991）Biochemistry 30，5546-5555]。为了解决这个问题，野生型（WT）和突变型（L36 I）flavocytochromes bi已在大肠杆菌中表达。这两种形式纳入血红素和FMN辅基和催化活性。突变L36 I是血红素结合缝隙内的保守取代，旨在改变血红素的中点电位（Em）以改变叶酸-FSQ/血红素平衡。电位滴定法得到WT和L36 I型的Em值（pH7.0，25 ℃）分别为+8和-28 mV.在不存在丙酮酸的情况下，WT和L36 I物质的FMN中点电位（-58 mV，= 2）在实验误差范围内相同，在存在丙酮酸的情况下也相同（+5 mV，n- 1）。这些结果表明FMN和血红素之间不存在氧化还原协同作用。电子受体铁氰化物（311和304 s-1，WT和L36 I）和细胞色素c（211和192 s-1，WT和L36 I）的转换数相似，表明FSQ对血红素电子转移缺乏速率限制。以细胞色素c作为受体的丙酮酸抑制在WT和L36 I形式中是竞争性的，并且与FSQ的丙酮酸连接的抑制不一致。2.3)催化L-乳酸氧化为丙酮酸，并将还原当量转移至两个细胞色素c分子（阿普尔比& Morton，1954）。该蛋白是四聚体，每个相同的亚基（57.5 kDa）含有一个FMN 1和一个血红素分子（Pajot & Groundinsky，1970;阿普尔比& Morton，1954）。最初，L-乳酸盐还原FMN，然后还原当量依次传递到
Revised Manuscript Received September 10, 1992 abstract: Kinetic and thermodynamic properties of yeast flavocytochrome bi (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 thermodynamicdriving force for electron transfer from FSQto heme. Pyruvateinhibits flavocytochrome bi, 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 bi have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. Themutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (£ m) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded Em values (pH 7.0, 25 C) of+ 8 and-28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV,= 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.Yeast flavocytochrome bi (L-lactate dehydrogenase, EC 1.1. 2.3) catalyzes the oxidation of L-lactate to pyruvate, with the transfer of reducing equivalents to two cytochrome c molecules (Appleby & Morton, 1954). The protein is tetrameric with each identical subunit (57.5 kDa) containing one FMN1 and one heme molecule (Pajot & Groundinsky, 1970; Appleby & Morton, 1954). Initially L-lactate reduces FMN, and reducing equivalents then pass sequentially to the
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通过改变黄素氧化还原电位来调节脱氢酶/单电子转移酶。
DOI：
10.1111/j.1432-1033.1986.tb09516.x
发表时间：
1986
期刊：
European journal of biochemistry
影响因子：
--
作者：
M. Tegoni;J. Janot;F. Labeyrie
通讯作者：
F. Labeyrie
影响因子：
56.9
作者：
ROSE, GD;GESELOWITZ, AR;ZEHFUS, MH
通讯作者：
ZEHFUS, MH
影响因子：
2.9
作者：
WOLFENDEN, R;ANDERSSON, L;SOUTHGATE, CCB
通讯作者：
SOUTHGATE, CCB
还原速率常数与氧化还原电位之间的相关性作为系统研究电子转移蛋白反应机制的基础。
DOI：
10.1073/pnas.80.22.6740
发表时间：
1983
影响因子：
11.1
作者：
Meyer,TE;Przysiecki,CT;Watkins,JA;Bhattacharyya,A;Simondsen,RP;Cusanovich,MA;Tollin,G
通讯作者：
Tollin,G
影响因子：
5.6
作者：
MCGREGOR, MJ;ISLAM, SA;STERNBERG, MJE
通讯作者：
STERNBERG, MJE