Gilderson Gwen, Salomonsson Lina, Aagaard Anna, Gray Jimmy, Brzezinski Peter, Hosler Jonathan
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Biochemistry. 2003 Jun 24;42(24):7400-9. doi: 10.1021/bi0341298.
The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.
细胞色素c氧化酶的催化核心由三个亚基组成，其中亚基I和II包含所有氧化还原活性金属中心，亚基III是一种七跨膜螺旋蛋白，与亚基I结合。亚基III的N端区域与亚基I的D132相邻，D132是D途径的初始质子受体，该途径将质子从蛋白质表面转移到约30埃远的埋藏活性位点。缺少亚基III只会略微改变氧化酶在pH 6.5时的初始稳态活性，但随着pH值升高，活性急剧下降，稳态O₂还原的表观pK(a)为7.2。当存在亚基III时，细胞色素氧化酶在较高pH下更具活性，稳态O₂还原的表观pK(a)为8.5。单周转实验表明，在pH 8时，通过D途径的质子摄取在存在亚基III时从>10000 s⁻¹减慢到不存在时的350 s⁻¹。在低pH（5.5）时，缺乏亚基III的氧化酶的D途径恢复了其快速摄取质子的能力。对F→O转变的分析表明，在不存在亚基III时，D途径的表观pK(a)为6.8，与稳态O₂还原的表观pK(a)（7.2）相似。由于D132的羧基将更多地暴露于溶剂水，其本身的pK(a)在不存在亚基III时可能会下降。或者，去除亚基III后，D途径的质子天线的一部分可能会丢失。有人提出，亚基III在正常氧化酶中的一个作用是在生理pH下维持通过D途径的快速质子摄取。
