Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Abstract

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron-and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from -3 ,us to several milliseconds, reflecting internal electrontransfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV =

Functional interactions between membrane-bound transporters and membranes

“…Results from earlier studies have shown that the PCET rate is independent of the composition of the buffer or its concentration (17,21), which indicates that buffer molecules do not act as proton donors or acceptors for the protons released through the K pathway. As the observed PCET rate represents the sum of the release and uptake rates, it is not straightforward to calculate a second-order proton-uptake rate constant.…”

“…1) and continues toward the catalytic site via a highly conserved Lys I 362. 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).…”

“…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). The proton donor is a water molecule at the catalytic site, which interacts electrostatically with heme a 3 .…”

“…Upon proton release, the formed hydroxide binds to the oxidized heme a 3 (21). Even though this proton release occurs in the opposite direction to that during turnover, the kinetics of the proton-coupled electron transfer (PCET) reflects both the proton uptake and release rates (the sum of these rates) through the K pathway (15,17). Formation of the mixed-valence-CO (from the oxidized form) was found to be accompanied by the uptake of approximately two protons, and this value was almost independent of pH (22).…”

Nanosecond electron tunneling between the hemes in cytochrome bo 3

“…This reaction was originally reported to consist of two phases (5,6): a Ϸ3-s phase of eT between the heme groups was followed by slower electron equilibration with an additional copper site (Cu A ) that lies at the membrane interface in some of the heme-copper oxidases but that is lacking in others (7,8). More recently, it was shown that a third phase of interheme eT (50 s to milliseconds) that is coupled to proton release from the binuclear center can be observed after CO photolysis at high pH (9)(10)(11). However, with the quinol-oxidizing cytochrome bo 3 from Escherichia coli, even more phases were discerned (12).…”