Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc(1) complex

by Engstrom, Gregory J.; Rajagukguk, Ray Honest Johnson; Saunders, Aleister J.; Patel, Chetan N.; Rajagukguk, Sany Clean Johnson; Merbitz-Zahradnik, Torsten; Xiao, Kunhong; Pielak, Gary J.; Trumpower, Bernard; Yu, Chang-An; Yu, Linda; Durham, Bill; Millett,

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc(1) to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)(2)(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru-z-39-Cc. The Ru-z-39-Cc derivative has the same steady-state activity with yeast cytochrome bc(1) as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru-z-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc(1) and Ru-z-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome cl to heme c with a rate constant of 1.4 x 10(4) s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc(1). The interaction of yeast Ru-39-Cc with yeast cytochrome bc(1) is stronger than that of horse Ru-39-Cc with bovine cytochrome bc(1), suggesting that nonpolar interactions are stronger in the yeast system.

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1520-4995; 0006-2960