Papa S, Capitanio N, Villani G, Capitanio G, Bizzoca A, Palese L L, Carlino V, De Nitto E
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Biochimie. 1998 Oct;80(10):821-36. doi: 10.1016/s0300-9084(00)88877-x.
In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanisms, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit I of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a3. One group is apparently linked to CuB. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and CuB in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H+/e- at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conversion of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit I of the aa3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.
在过去几年中，越来越多的证据支持质子泵在细胞色素系统中的协同模型（矢量玻尔机制）适用于血红素 - 铜氧化酶。矢量玻尔机制基于与金属中心氧化还原转变相关的短程和长程质子动力协同效应。氧化型和还原型牛心细胞色素c氧化酶的晶体结构显示，在还原时，氧化酶亚基I中会发生长程构象变化。细胞色素c氧化酶晶体结构分析表明，存在由氨基酸残基组成的氢键网络，这些氨基酸残基可能会发生与氧化还原相关的pK值变化，从而导致跨膜质子转运。我们的研究小组已经确定了四个经历可逆氧化还原相关pK值变化的蛋白水解基团。其中两个基团与血红素a3的氧化还原转变有关。一个基团显然与铜B有关。第四个基团与血红素a的氧化还原有关。我们已经表明，牛氧化酶中与血红素a和铜B相关的氧化还原玻尔效应导致的质子转移表现出膜矢量不对称性，即还原时质子从内部水相空间（N）摄取，金属氧化时质子释放到外部空间（P）。这种质子摄取和释放的方向正是矢量玻尔机制所预期的。与血红素a相关的基团在接近中性的pH值下每电子可转移多达0.9个H +，可为质子泵提供主要贡献。有人提出，与通过血红素a的电子流相关的泵送质子的转运利用了一个通道（通道D），该通道从N端的一个保守天冬氨酸延伸到位于血红素a和双核中心之间的一个保守谷氨酸。在通过血红素a进行电子流时，这个谷氨酸的羧基将泵送质子输送到P相，一旦从N相重新质子化，随后移动以将化学质子输送到双核中心，这些质子在氧化还原循环中用于将过氧化物转化为高铁血红素以及将后者转化为氧中间体。将枯草芽孢杆菌aa3 - 600喹啉氧化酶亚基I中的质子化残基定点突变为非极性残基表明，保守的赖氨酸304对氧化酶的质子泵送活性至关重要。细胞色素c氧化酶的晶体结构表明，该赖氨酸位于一个通道的N端，该通道转运用于生成过氧化物中间体所消耗的质子。通过替换赖氨酸来抑制该途径，会使质子从通道D短路到双核中心，在那里质子用于氧还原的化学反应。
