Smirnova I A, Adelroth P, Gennis R B, Brzezinski P
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Biochemistry. 1999 May 25;38(21):6826-33. doi: 10.1021/bi982865j.
The aspartate-132 in subunit I (D(I-132)) of cytochrome c oxidase from Rhodobacter sphaeroides is located on the cytoplasmic surface of the protein at the entry point of a proton-transfer pathway used for both substrate and pumped protons (D-pathway). Replacement of D(I-132) by its nonprotonatable analogue asparagine (DN(I-132)) has been shown to result in a reduced overall activity of the enzyme and impaired proton pumping. The results from this study show that during oxidation of the fully reduced enzyme the reaction was inhibited after formation of the oxo-ferryl (F) intermediate (tau congruent with 120 microseconds). In contrast to the wild-type enzyme, in the mutant enzyme formation of this intermediate was not associated with proton uptake from solution, which is the reason the DN(I-132) enzyme does not pump protons. The proton needed to form F was presumably taken from a protonatable group in the D-pathway (e.g., E(I-286)), which indicates that in the wild-type enzyme the proton transfer during F formation takes place in two steps: proton transfer from the group in the pathway is followed by faster reprotonation from the bulk solution, through D(I-132). Unlike the wild-type enzyme, in which F formation is coupled to internal electron transfer from CuA to heme a, in the DN(I-132) enzyme this electron transfer was uncoupled from formation of the F intermediate, which presumably is due to the impaired charge-compensating proton uptake from solution. In the presence of arachidonic acid which has been shown to stimulate the turnover activity of the DN(I-132) enzyme (Fetter et al. (1996) FEBS Lett. 393, 155), proton uptake with a time constant of approximately 2 ms was observed. However, no proton uptake associated with formation of F (tau congruent with 120 micros) was observed, which indicates that arachidonic acid can replace the role of D(I-132), but it cannot transfer protons as fast as the Asp. The results from this study show that D(I-132) is crucial for efficient transfer of protons into the enzyme and that in the DN(I-132) mutant enzyme there is a "kinetic barrier" for proton transfer into the D-pathway.
球形红杆菌细胞色素c氧化酶亚基I中的天冬氨酸-132（D（I-132））位于蛋白质的细胞质表面，处于用于底物和泵送质子的质子转移途径（D途径）的入口点。已表明用其不可质子化类似物天冬酰胺（DN（I-132））替代D（I-132）会导致该酶的总体活性降低以及质子泵送受损。本研究结果表明，在完全还原的酶氧化过程中，反应在氧代高铁（F）中间体形成后受到抑制（τ约为120微秒）。与野生型酶不同，在突变酶中，该中间体的形成与从溶液中摄取质子无关，这就是DN（I-132）酶不泵送质子的原因。形成F所需的质子大概取自D途径中的一个可质子化基团（例如，E（I-286）），这表明在野生型酶中，F形成过程中的质子转移分两步进行：首先是质子从途径中的基团转移，然后是通过D（I-132）从本体溶液中更快地重新质子化。与野生型酶不同，在野生型酶中F的形成与从CuA到血红素a的内部电子转移偶联，在DN（I-132）酶中，这种电子转移与F中间体的形成解偶联，这大概是由于从溶液中摄取电荷补偿质子受损所致。在存在花生四烯酸的情况下，已表明花生四烯酸可刺激DN（I-132）酶的周转活性（费特等人（1996年）《欧洲生物化学学会联合会快报》393，155），观察到质子摄取的时间常数约为2毫秒。然而，未观察到与F形成相关的质子摄取（τ约为120微秒），这表明花生四烯酸可以替代D（I-132）的作用，但它不能像天冬氨酸那样快速转移质子。本研究结果表明，D（I-132）对于质子有效转移到酶中至关重要，并且在DN（I-132）突变酶中，质子转移到D途径存在“动力学障碍”。
