Individual heme a and heme a 3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a , chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa -type cytochrome c oxidase consists of overlapping bands of the hemes a and a . It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a and a in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a and a using two approaches. First, we reconstructed bovine CcO heme a spectrum using a selective Ca -induced spectral shift of the heme a . Second, we investigated photobleaching of the reduced Thermus thermophilus ba - and bovine aa -oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B -, B -transitions of both reduced hemes. The heme a spectrum is shifted to the red relative to heme a spectrum. The ∼425 nm shoulder is mostly attributed to heme a .