Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17661-6. doi: 10.1073/pnas.1107543108. Epub 2011 Oct 12.
Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O(2) reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb(3)) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb(3) numbering) in the accessory subunit CcoP (E25(P)). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb(3) by O(2) is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25(P) as the entry point to the proton pathway in cbb(3) and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O(2) reduction. The cbb(3) is in addition to O(2) reduction capable of NO reduction, an activity that was largely retained in the E25(P) variants, consistent with a scenario where NO reduction in cbb(3) uses protons from the periplasmic side of the membrane.
血红素铜氧化酶（HCuOs）是线粒体和许多细菌呼吸链的最后一个组成部分。它们催化 O2 还原，并将其与膜内质子动力的维持相偶联。在类似于线粒体的 A 家族 HCuOs 中，有两条已建立的质子转移途径从细胞质通向活性位点，即 D 途径和 K 途径。在 C 家族（cbb(3)）HCuOs 中，最近的研究表明仅使用一条途径，类似于 K 途径。在这项工作中，我们研究了该途径建议入口点（Rhodobacter sphaeroides cbb(3)编号为 Glu-25）在辅助亚基 CcoP（E25(P)）中的功能重要性。我们表明，在缺乏可质子化的 Glu-25 的变体中，催化周转率严重减慢。此外，当 Glu-25 被 Ala 或 Gln 取代时，在完全还原的 cbb(3)被 O2 氧化过程中从溶液中摄取质子会受到特异性和严重的损害，其速率常数比野生型慢 100-500 倍。因此，我们的结果支持 E25(P)作为 cbb(3)质子途径进入点的作用，并且该途径是主要的质子途径。这与 A 型 HCuOs 形成对比，在 A 型 HCuOs 中，D（而不是 K）途径在 O2 还原过程中被使用。cbb(3)除了能够还原 O2 之外，还能够还原 NO，这一活性在 E25(P)变体中基本保留，这与在 cbb(3)中还原 NO 使用膜周质侧质子的情况相符。
