Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen

The reduction of dioxygen to water by cytochrome c oxidase was monitored in the Soret region following photolysis of the fully reduced CO complex. Tim e-resolved optical absorption difference spectra collected between 373 and 521 nm were measured at delay times from 50 ns to 50 ms and analyzed using singular value decomposition and multiexponential fitting. Five processes w ere resolved with apparent lifetimes of 0.9 mu s, 8 mu s, 36 mu s, 103 mu s , and 1.2 ms. A mechanism is proposed and spectra of intermediates are extr acted and compared to model spectra of the postulated intermediates. The mo del builds on an earlier mechanism that used data only from the visible reg ion (Sucheta et al. (1997) Biochemistry 36, 554-565) and provides a more co mplete mechanism that fits results from both spectral regions. Intermediate 3, the ferrous-oxy complex (compound A) decays into a 607 nm species, gene rally referred to as P, which is converted to a 580 nm ferryl form (F-o) on a significantly faster time scale. The equilibrium constant between P and F-o is 1. We propose that the structure of P is a(3)(4+)=O Cu-B(2+)-OH- Wit h an oxidizing equivalent residing on tyrosine 244, located close to the bi nuclear center. Upon conversion of P to F-o, cytochrome a donates an electr on to the tyrosine radical, forming tyrosinate. Subsequently a proton is ta ken up by tyrosinate, forming F-I [a(3)(4+)=O Cu-B(2+)-OH- a(3+) Cu-A(+)]. This is followed by rapid electron transfer from Cu-A to cytochrome a to pr oduce F-II [a(3)(4+)=O Cu-B(2+)-OH- a(2+) Cu-A(2+)].