Pawate Ashtamurthy S, Morgan Joel, Namslauer Andreas, Mills Denise, Brzezinski Peter, Ferguson-Miller Shelagh, Gennis Robert B
Center for Biophysics and Computational Biology and Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2002 Nov 12;41(45):13417-23. doi: 10.1021/bi026582+.
The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.
血红素铜氧化酶将氧气还原为水过程中释放的自由能转化为跨膜质子电化学势（质子动力）。该酶的一个基本结构元件是D通道，它被认为是质子的输入途径，这些质子既用于形成水的质子（“化学质子”），也用于跨脂质膜转运的质子（“泵出质子”）。D通道包含一串水分子，从靠近细胞质（“内侧”）酶表面的天冬氨酸（球形红细菌氧化酶中的D132）延伸约25埃至蛋白质内部的谷氨酸（E286）。这些酸性残基中的任何一个被其相应的酰胺取代（D132N或E286Q）的突变都会导致酶活性的严重抑制。在当前的工作中，D通道中的一个天冬酰胺被相应的酸取代（N139突变为D；牛酶中的N98），产生了显著的后果。N139D突变不仅完全消除了质子泵出，同时还使稳态细胞色素氧化酶活性大幅增加（150 - 300%）。通过检查催化循环中各个步骤的速率，对球形红细菌氧化酶的N139D突变体进行了进一步表征。在厌氧条件下，与氧气反应之前，完全氧化的酶中血红素a3的还原速率与野生型氧化酶相同，且未加速。然而，如更快速的F→O转变所示，完全还原的酶与氧气的反应速率因N139D突变而加快。虽然氧化中间体的形成和衰减速率发生了改变，但氧化中间体的性质并未因N139D突变而受到干扰。
