Yi Sophia M, Taguchi Alexander T, Samoilova Rimma I, O'Malley Patrick J, Gennis Robert B, Dikanov Sergei A
§Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
†Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
Biochemistry. 2015 Aug 18;54(32):5030-44. doi: 10.1021/acs.biochem.5b00528. Epub 2015 Aug 6.
Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.
细胞色素aa3-600是电子传递途径中的一种末端氧化酶，通过主动泵出质子来促进电化学膜电位的形成。这种酶复合物的一个显著特征是，在将双氧还原为水时，它使用甲萘醌作为电子供体，而不是细胞色素c。该酶在一个对催化作用很重要的高亲和力位点稳定甲萘醌半醌自由基（SQ）。与半醌相互作用的残基之一是Arg70。我们制备了R70H突变体，并通过先进的X波段和Q波段电子顺磁共振对甲萘醌半醌自由基进行了表征。R70H突变体结合的SQ与一个氢键结合的氮表现出强烈的各向同性超精细耦合（a(14)N≈2.0 MHz）。基于其四极耦合常数e(2)qQ/h = 1.44 MHz（质子化咪唑氮的典型值），这个氮来自组氨酸侧链。在野生型细胞色素aa3-600中，SQ反而与Arg70侧链的Nε形成氢键。对(1)H二维电子自旋回波包络调制（ESEEM）光谱的分析表明，该突变也改变了SQ与周围蛋白质之间氢键的数量和强度。尽管SQ的直接环境发生了改变，但R70H突变体仍保持催化活性。这些发现与密切同源物——大肠杆菌的细胞色素bo3泛醇氧化酶中的等效突变形成对比，在那里R71H突变消除了功能。
