Strong Excitonic Interactions in the Oxygen-Reducing Site of bd-Type Oxidase: The Fe-to-Fe Distance between Hemes d and b595 is 10 Å

Abstract

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A. A., and Gennis, R. B. (2001) Biochemistry 40, 8548−8556]. The absorption bands of ferrous heme b₅₉₅ are absent from the spectrum of the dithionite-reduced E445A form of cytochrome bd. The difference between the spectra of the dithionite-reduced WT and E445A enzymes indicates that in the mutant, heme b₅₉₅ is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b₅₉₅ or heme b₅₅₈. The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b₅₉₅, and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b₅₉₅ remains in the ferric state. Modeling the excitonic interactions in both absorption and CD spectra has been carried out, yielding an estimate of the Fe-to-Fe distance between heme d and heme b₅₉₅ of about 10 Å. The physical proximity supports the hypothesis that heme d and heme b₅₉₅ can form a di-heme oxygen reducing site, a unique structure for respiratory oxidases.