Electrogenic events upon photolysis of CO from fully reduced cytochrome c oxidase

Abstract

CO photolysis from fully reduced Paracoccus denitrificans aa3-type cytochrome c oxidase in the absence of O2 was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5μs) phase was followed by slower potential generation with a time constant varying from 8μs at pH 7 to 250μs at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10–30%. The amplitude of the fast phase corresponds to charge movement of 0.83Å perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of CcO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.