Q-band Electron Nuclear Double Resonance (ENDOR) and X-band EPR of the Sulfobetaine 12 heat-teated Cytochrome c Oxidase Complex

Siegfried M. Musser, Yang-Cheng Fann, Ryszard J. Gurbiel, Brian M. Hoffman and Sunney I. Chan
J. Biol. Chem. 272 202-209 (1997)

SUMMARY: Heat treatment of the bovine cytochrome c oxidase complex in the zwitterionic detergent sulfobetaine 12 (SB-12) results in loss of subunit III and the appearance of a Type II copper center, as characerized by EPR spectroscopy. Previous authors (Nilsson et al (1988) Biochemistry 27, 8254-8260) have interpreted this type II Cu center as a modified version of the Cu_A site. By using electron nuclear double resonance spectroscopy it is found that that the Cu_A proton and nitrogen resonances remain present in the SB-12 heat-treated enzyme. and that 3 new nitrogen coupling constants appear having hyperfine coupling constants consistent with histidine ligation. These hyperfine coupling constants correlate well with those found recenly for the Cu_B histidines of the cytochrome aa3-600 quinol oxidase from Bacillus subtilis (Fann et al (1995) Biochemistry 34, 10245-10255). In addition, the total EPR-detectable type II copper concentration per enzyme molecule is virtually indistinguishable from the EPR spectrum of Cu_B of the as-isolated cytochrome bo₃ from E. coli. These data indicate that the type II copper sdpecies that apppears results from a breaking of the styrong antiferromagnetic coupling of the heme a₃-Cu_B binuclear center.