EPR characterization of the cytochrome b- c1 complex from Rhodobacter sphaeroides

Abstract

EPR characteristics of cytochrome c 1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b- c 1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c 1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b- c 1 complex are: g x = 1.8 with a small shoulder at g = 1.76, g y = 1.89 and g z = 2.02, similar to those observed with the mitochondrial enzyme. The g x = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the g z and and shifts it downfield from g = 2.02 to 2.03, and shifts g x upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of g y but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the g y from 1.89 to 1.94 and broadens the g x signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the g y and g z signals, but they broaden and shift the g x signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b- c 1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV. The concentration of antimycin sensitive Q-radical correlates well with the enzymatic activity of the complex. The antimycin-sensitive ubisemiquinone radical exhibits a pH dependency of approx. 60 mV / pH unit, between pH 8.0 and 9.0, suggesting the possible involvement of a dissociable group, with a pK a between 8.0 and 9.0, in Q-binding.