Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies.

Abstract

The cytochrome bo complex is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. To clarify the structural differences of the binuclear reaction center between the cytochrome bo complex and the mitochondrial cytochrome c oxidase, a combined study using EPR and Fourier transform infrared spectroscopies was carried out. The EPR spectrum of the highly purified cytochrome bo complex in the air-oxidized state showed a broad EPR signal (peak g* = 3.7) from an integer spin system. This confirms the existence of the spin-spin exchange-coupled binuclear site, in which the Feo3+ and CuB2+ centers were bridged by an unknown ligand (X). Binding of azide at the binuclear site as an ionic modulator weakened the strength of the spin-spin exchange coupling and thus caused a narrowing of the broad EPR signal. Binding of another modulator, formate, at the binuclear site caused the formation of EPR signals at g' = 12 and 2.7, which are very similar to those observed for cytochrome c oxidase. Cyanide replaced the bridging ligand (X) to form an Feo(3+)-C-N-CuB2+ structure in which strong spin-spin exchange coupling is expected, leading to a complete EPR-invisible state. Infrared evidence (a 2146 cm-1 C-N stretching band for the cyanide complex and a 2041 cm-1 azide antisymmetric stretching band for the azide complex) supported the theory that these ligands form bridging structures at the binuclear center, as previously observed for cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)