White P, Manson F D, Brunt C E, Chapman S K, Reid G A
Department of Chemistry, University of Edinburgh, Scotland, U.K.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):89-94. doi: 10.1042/bj2910089.
The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.
黄素细胞色素b2（L-乳酸：细胞色素c氧化还原酶）的两个不同结构域由一个典型的铰链肽连接。酿酒酵母和异常汉逊酵母的黄素细胞色素b2中，这个结构域间铰链的氨基酸序列存在显著差异。据信，这种铰链差异导致了两种酶动力学性质的不同。为探究铰链的重要性，构建了一种种间杂交酶，它包含酿酒酵母酶的大部分，但含有异常汉逊酵母黄素细胞色素b2的铰链。通过稳态和停流方法研究了这种“铰链交换”酶的动力学性质。从以铁氰化物为受体的稳态实验（活性仅比野生型酶低3倍）以及监测黄素还原的停流实验（比野生型酶慢2.5倍）可以明显看出，铰链交换酶仍然是一种良好的乳酸脱氢酶。铰链交换突变的主要影响是显著降低了该酶作为细胞色素c还原酶的效率；细胞色素c还原的催化常数（kcat.）下降了100多倍，从野生型酶在25℃、pH 7.5时的207±10 s-1降至突变型酶的1.62±0.41 s-1。细胞色素c还原酶活性的这种下降是由于FMN和血红素基团之间结构域间电子传递不佳所致。这可以通过以下事实得到证明：铰链交换酶中血红素还原的催化常数（通过停流法测量）为1.61±0.42 s-1，与细胞色素c还原的值相同，比野生型酶的值低约300倍。从这些以及其他动力学参数，包括用[2-2H]乳酸进行的动力学同位素效应，我们得出结论，铰链在允许黄素细胞色素b2的两个结构域之间进行高效电子传递方面起着关键作用。
