Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.
Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.
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细胞色素氧化酶的质子易位化学计量：使用快速响应的氧电极。
DOI：
10.1073/pnas.79.23.7218
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发表时间：
1982
影响因子：
11.1
通讯作者：
Lehninger,AL
中科院分区：
文献类型：
--
作者：
Reynafarje,B;Alexandre,A;Davies,P;Lehninger,AL
通过测量在K+（含valinomycin）存在下的初始电子流速率和H+喷射速率，确定了大鼠肝有丝分裂体中加入铁细胞色素c的细胞色素氧化酶（EC 1.9.3.1）向氧的电子传递耦合载体H+喷射的机制化学计量学。使用了三种不同的电子流测量方法：(a)铁细胞色素c氧化的双波长分光光度法，(b)在质子团存在下吸收标量H+还原O2，以及(c)快速响应的无膜氧电极。速率测量的可靠性首先根据已知的细胞色素氧化酶（2铁细胞色素c + 2H+ + 1/2O2导致2铁细胞色素c + H2O）在过量质子载体存在下的标量反应的化学计量学建立。三种方法直接观察到的K+ +/O射血比均显著大于3.0。然而，由于喷射出的H+回流到有丝质体中的速率非常高，并且随着膜上产生的δ pH的增加而增加，因此从反应开始观察到的H+/O比率就会迅速下降。利用快速响应的无膜氧电极对有丝分裂体氧化铁细胞色素c的动力学分析表明，该反应在O2中是一级的，并且可以准确地外推O2摄取和H+排出的速率到零时间。此时，细胞膜上的pH值为零，细胞色素氧化酶反应的H+/O喷射比，由零时的速率得到，接近4.0。
The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.