Single Mutations That Redirect Internal Proton Transfer in the ba3 Oxidase from Thermus thermophilus

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• 作者：Irina Smirnova ; Hsin-Yang Chang ; Christoph von Ballmoos ; Pia 脛delroth ; Robert B. Gennis ; Peter Brzezinski
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：October 8, 2013
• 年：2013
• 卷：52
• 期：40
• 页码：7022-7030
• 全文大小：368K
• 年卷期：v.52,no.40(October 8, 2013)
• ISSN：1520-4995

文摘

The ba₃-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa₃-type oxidases proton uptake to the catalytic site and 鈥減ump site鈥?occurs simultaneously. However, with ba₃ oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa₃ oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba₃ CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba₃ CytcO uses only a single proton pathway for transfer of the substrate and 鈥減umped鈥?protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.