Baserga Federico, Dragelj Jovan, Kozuch Jacek, Mohrmann Hendrik, Knapp Ernst-Walter, Stripp Sven T, Heberle Joachim
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany.
Macromolecular Modelling Group, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
Front Chem. 2021 Apr 27;9:669452. doi: 10.3389/fchem.2021.669452. eCollection 2021.
Cytochrome oxidase (CO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CO from . The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (RCO, RCO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from RCO to RCO. We performed potential jump experiments to accumulate the RCO and RCO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a. Our experiments also suggest propionate A of heme a changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.
细胞色素氧化酶（CO）是一种跨膜蛋白复合物，它将分子氧还原为水，同时将质子跨线粒体内膜转运。其辅因子氧化还原状态的变化触发了氧的还原和矢量质子转移，其中包括一个尚未确定的质子加载位点。在这项工作中，我们利用一氧化碳（CO）作为来自[具体来源未明确]的CO双核中心的振动斯塔克效应（VSE）探针。使用傅里叶变换红外（FTIR）吸收光谱电化学法监测CO伸缩频率随电势的变化。我们观察到三种不同的氧化还原状态（RCO、RCO和O），确定了它们的中点电势，并将所得电场与静电计算结果进行比较。得出了由RCO到RCO的氧化还原转变引起的局部电场强度变化为+2.9 MV/cm。我们进行了电势跃变实验以积累RCO和RCO物种，并研究了蛋白质指纹区域的FTIR差光谱。实验结果与计算结果的比较表明，关键的谷氨酸残基E286在观察到的状态下被质子化，并且在血红素a的氧化还原转变时其氢键环境受到干扰。我们的实验还表明，血红素a的丙酸A与第二个辅因子血红素a的氧化还原状态协同改变其质子化状态。这支持了丙酸侧链作为质子加载位点一部分的作用。
