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

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have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A. A., and Gennis, R. B. (2001) Biochemistry 40, 8548-8556].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 b(595) 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 b(595) is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b(595) or heme b(558). The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b(595), and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b(595) 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 b(595) of about 10 angstrom. The physical proximity supports the hypothesis that heme d and heme b(595) can form a di-heme oxygen reducing site, a unique structure for respiratory oxidases.