Internal Electron Transfer in Cytochrome c Oxidase from Rhodobacter sphaeroides

Abstract

Absorbance changes following CO dissociation by flash photolysis from mixed-valence aa3 cytochrome oxidase from Rhodobacter sphaeroides have been followed in the Soret and alpha regions. They reflect internal electron transfer in the partially reduced enzyme, and the kinetics of the reactions has been determined. As with the bovine enzyme, three kinetic phases are found with relaxation time constants at neutral pH of about 3 microseconds, 35 microseconds, and 1 ms. The first reaction phase represents electron transfer from cytochrome a3 to cytochrome a, and the extent of this reaction is about 3 times larger compared to the bovine enzyme. The energetics of the reaction has been analyzed on the basis of measurements of its temperature dependence. The reorganization energy is close to 120 kJ mol-1, and it is suggested that this rather high value is the result of changes in solvation at the cytochrome a3-CuB site. The subsequent electron transfer between cytochrome a and CuA, with a time constant of 35 microseconds, is almost activationless and has a very low reorganization energy. The final phase, with a time constant close to 1 ms at neutral pH, represents a further shift in the equilibrium between cytochrome a3 and cytochrome a, and it is limited by proton-transfer reactions. The pKa values of the groups involved are significantly shifted in the bacterial oxidase compared to the bovine one. The total extent of electron transfer in the three backflow reactions has also been determined by a comparison of the CO-recombination rates in the mixed-valence and fully reduced enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the Electron Redistribution in Mixed Valence Cytochrome c Oxidase by Protein Conformational Changes

“…It is followed by electron transfer from heme a to Cu A at about 2 ϫ 10 4 s Ϫ1 . Similar studies have been carried out on other members of the terminal oxidase super family, including those from the bacterium Rhodobacter sphaeroides and cytochrome bo 3 from Escherichia coli (12,14,15), and the electron transfer events in these enzymes were detected on similar time scales. Recently, the rates and the extent of the electron transfer were questioned by Verkhovsky et al (16), who reported that an electron transfer event between the two hemes occurs on a time scale much faster than 100 ns, the limit of their instrumental resolution, and accounted for about 50% amplitude of the total amount of electron transfer from heme a 3 to heme a.…”

“…The first phase is assigned to the electron transfer from heme a 3 to heme a, which occurs at a rate of ϳ3 ϫ 10 5 s Ϫ1 (8,12,13). It is followed by electron transfer from heme a to Cu A at about 2 ϫ 10 4 s Ϫ1 .…”

Functional interactions between membrane-bound transporters and membranes

“…This behavior was attributed to a titration of an internal proton donor/ acceptor within the K pathway (18,41). Because this maximum rate is slower than that observed with the membrane-reconstituted wild-type CytcO at pH 9.5, the effect of the membrane cannot be a simple pK a shift of this proton donor.…”

“…Results from earlier studies have shown that the rate of the PCET in detergent solution is pH dependent with a maximum value of ∼2;000 s −1 (τ ¼ 500 μs) at low pH (<7) (18). This behavior was attributed to a titration of an internal proton donor/ acceptor within the K pathway (18,41).…”

“…Proton transfer through this pathway can be studied time resolved upon flash photolysis of a carbon monoxide (CO) ligand from the heme a 3 iron in CytcO in which the catalytic site is reduced while heme a and Cu A are oxidized (15,17) (often referred to as the mixed-valence CytcO). Because the CO ligand stabilizes the reduced state of heme a 3 , after flash photolysis of the complex the electron initially residing at heme a 3 is transferred to heme a over time scales <3 μs (18)(19)(20). On a slower time scale, there is further electron transfer from heme a 3 to heme a (and to some extent also Cu A ), which is coupled to proton release from the catalytic site to solution on the n side of the membrane, via the K pathway, with a time constant of ∼1 ms (17).…”