Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.
摘要
本文描述了一些技术和实验，这些技术和实验涉及还原型细胞色素c以及在经各种试剂还原后再用氧气或铁氰化物重新氧化时，细胞色素c氧化酶中EPT可检测到的毫秒级动力学变化。还报道了一些在配体存在下的实验。通过低温反射光谱监测光吸收。2. 在细胞色素c还原细胞色素c氧化酶的快速阶段（小于50毫秒），每个血红素a约0.5电子当量主要转移到细胞色素c氧化酶的低自旋血红素组分，部分转移到EPR可检测到的铜。在缓慢阶段（小于1秒），铜被重新氧化，高自旋铁血红素信号出现，主要为菱形组分。同时，655纳米处的吸收带降低，444纳米处的Soret带出现在还原型细胞色素a的分裂Soret带（442和447纳米）之间。3. 用氧气重新氧化还原型酶时，所有EPR和光学特征在6毫秒内恢复。在过量还原型细胞色素c存在下用氧气重新氧化时，可以观察到低自旋血红素和铜信号基本不存在但655纳米处吸收最大的状态，这表明低自旋血红素和铜组分在底物侧，而系统中655纳米吸收所代表的组分在氧气侧。用铁氰化物重新氧化时，655纳米吸收不容易恢复，但会积累由强菱形信号代表的高铁高自旋血红素。4. 用氧气重新氧化部分还原的酶时，菱形高自旋信号在6毫秒内消失，而轴向信号消失得更慢，这表明这些物种没有快速平衡。当部分还原的酶与CO混合时也有类似观察结果。5. 讨论了本文及随附论文的结果，并在此基础上提出了主要EPR信号和655纳米吸收的归属，其本质与先前发表的结果（Hartzell, C.R., Hansen, R.E.和Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477 - 2481）相同。低自旋（g = o; 2.2; 1.5）和缓慢出现的高自旋（g = 6; 2）信号都归因于高铁细胞色素a，而655纳米吸收被认为是由高铁细胞色素a3产生的，当它以与EPR不可检测的铜相互作用的状态存在时。讨论了该提议的其他可能性和可能存在的不一致之处。
