EPR characterization of the cytochrome b-c1 complex from Rhodobacter sphaeroides.
EPR characteristics of cytochrome c1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b-c1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c1 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-c1 complex are: gx = 1.8 with a small shoulder at g = 1.76, gy = 1.89 and gz = 2.02, similar to those observed with the mitochondrial enzyme. The gx = 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 gz and and shifts it downfield from g = 2.02 to 2.03, and shifts gx upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of gy but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the gy from 1.89 to 1.94 and broadens the gx signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the gy and gz signals, but they broaden and shift the gx 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-c1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
McCurley
J P
JP
Department of Biochemistry, OAES, Oklahoma State University, Stillwater 74078.
Miki
T
T
Yu
L
L
Yu
C A
CA
D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D003067:Coenzymes; D003573:Cytochrome b Group / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008689:Methacrylates; D010084:Oxidation-Reduction; D012242:Rhodobacter sphaeroides / Q000187:drug effects / Q000201:enzymology*; D013844:Thiazoles / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism / Q000031:analogs & derivatives
C025993:5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole; D003067:Coenzymes; D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; D008689:Methacrylates; D013844:Thiazoles; C032456:antimycin; D014451:Ubiquinone; D000968:Antimycin A; C030517:myxothiazol; D014450:Electron Transport Complex III; C024989:coenzyme Q10