ENDOR and ESEEM studies of cytochrome c oxidase: evidence for exchangeable protons at the CuA site.
ENDOR and ESEEM studies of cytochrome c oxidase: evidence for exchangeable protons at the CuA site.
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细胞色素 c 氧化酶的 ENDOR 和 ESEEM 研究：CuA 位点可交换质子的证据。
DOI：
10.1021/bi00212a042
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Babcock,GT
中科院分区：
文献类型：
--
作者：
Hansen,AP;Britt,RD;Klein,MP;Bender,CJ;Babcock,GT
用电子核双共振（ENDOR）和电子自旋回波包络调制（ESEEM）技术研究了细胞色素c氧化酶中Cua周围蛋白质环境中的质子是否易受溶剂交换的影响。将酶在静止或周转条件下在缓冲的D2 O中孵育90分钟，然后冷冻以淬灭氢/氘交换过程。氘代样品的ENDOR光谱与对照样品的ENDOR光谱基本相同。然而，ESEEM光谱提供了在缓冲D20中孵育后将氘引入CuA环境的明确指示。氘掺入的程度不受酶营业额。对ESEEM数据的分析表明，水与CuA位点相当接近，但不在金属的直接配位范围内。我们估计Cua中心和蛋白质/水界面之间的最小距离为5.4 μ m。这种相对短的表面分离距离与Cua在细胞色素氧化酶中作为细胞色素c的直接氧化剂的作用一致（Hill，B. C.（1991）J.Biol.Chem.266，2219-2226）。
Electron nuclear double resonance(ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used tostudy whether protons in the immediate protein environment around Cua in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D20. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the Cua site, but not in the immediate coordination sphere of the metal (s). We estimate a minimum distance of 5.4 Á between the Cua center and the protein/water interface. This relatively short surface separation distance is consistent with the role of Cua as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C.(1991) J. Biol. Chem. 266, 2219-2226).