Rotsaert Frederik A J, Covian Raul, Trumpower Bernard L
Department of Biochemistry, Dartmouth Medical School, 7200 Vail, Hanover, New Hampshire 03755, USA.
Biochim Biophys Acta. 2008 Mar;1777(3):239-49. doi: 10.1016/j.bbabio.2007.08.005. Epub 2007 Sep 6.
We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b(H) heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.
我们研究了酵母细胞色素bc1复合物变体中b血红素的稳态前动力学和热力学性质，这些变体在醌还原酶位点（中心N）发生了突变。色氨酸-30是一个高度保守的残基，与高电位b血红素（bH血红素）上的丙酸酯形成氢键。用半胱氨酸取代（W30C）会降低血红素的氧化还原电位，一个明显的结果是中心N处喹啉和血红素之间的电子转移速率降低。亮氨酸-198也紧邻b(H)血红素，L198F突变改变了血红素的光谱性质，但对其氧化还原性质或中心N处的电子转移动力学只有轻微影响。用谷氨酰胺或谷氨酸取代甲硫氨酸-221会导致稳定醌配体的疏水相互作用丧失。丝氨酸-20和谷氨酰胺-22形成了一个氢键网络，其中包括组氨酸-202、泛醌环的一个羰基和一个活性位点水。S20T突变对中心P有远程结构影响，对两个b血红素都有热力学影响。其他突变（M221E、M221Q、Q22E和Q22T）不影响中心P处泛醇氧化动力学，但会不同程度地改变中心N处的电子转移反应。稳态前还原动力学表明，这些突变改变了中心N处醌配体的结合，可能是通过扩大结合口袋，从而增加底物与bH血红素之间的距离。这些结果表明，可以区分结构和热力学因素对中心N功能的贡献。
