Geren Lois, Durham Bill, Millett Francis
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas, USA.
Methods Enzymol. 2009;456:507-20. doi: 10.1016/S0076-6879(08)04428-5.
Ruthenium photoreduction methods are described to study electron transfer from cytochrome c to cytochrome c oxidase and within cytochrome oxidase. Methods are described to prepare a ruthenium cytochrome c derivative Ru-39-Cc, by labeling the single sulfhydryl group on horse K39C with (4-bromomethyl-4'methylbipyridine) (bis-bipyridine)ruthenium(II). The ruthenium complex attached to Cys-39 on the opposite side of the heme crevice does not interfere with the interaction with cytochrome oxidase. Laser flash photolysis of a 1:1 complex between Ru-39-Cc and bovine cytochrome oxidase results in photoreduction of heme c within 1 microsec, followed by electron transfer from heme c to Cu(A) in cytochrome oxidase with a rate constant of 60,000 s(-1) and from Cu(A) to heme a with a rate constant of 20,000 s(-1). A new ruthenium dimer, Ru(2)Z, has been developed to reduce Cu(A) within 1 microsec with a yield of 60%, followed by electron transfer from Cu(A) to heme a and then to the heme a(3)/Cu(B) binuclear center. Methods are described to measure the single-electron reduction of each of the intermediates involved in reduction of oxygen to water by cytochrome oxidase, including P(m), F, O(H), and E.
描述了钌光还原方法，用于研究从细胞色素c到细胞色素c氧化酶以及细胞色素氧化酶内部的电子转移。描述了通过用(4-溴甲基-4'-甲基联吡啶)(双-联吡啶)钌(II)标记马K39C上的单个巯基来制备钌细胞色素c衍生物Ru-39-Cc的方法。附着在血红素裂隙另一侧Cys-39上的钌配合物不会干扰与细胞色素氧化酶的相互作用。Ru-39-Cc与牛细胞色素氧化酶之间1:1复合物的激光闪光光解导致血红素c在1微秒内光还原，随后电子以60,000 s(-1)的速率常数从血红素c转移到细胞色素氧化酶中的Cu(A)，并以20,000 s(-1)的速率常数从Cu(A)转移到血红素a。已开发出一种新的钌二聚体Ru(2)Z，可在1微秒内以60%的产率还原Cu(A)，随后电子从Cu(A)转移到血红素a，然后转移到血红素a(3)/Cu(B)双核中心。描述了测量细胞色素氧化酶将氧气还原为水过程中涉及的每种中间体单电子还原的方法，包括P(m)、F、O(H)和E。
