Ultrafast spectroscopy of electron transfer between hemes in bovine heart cytochrome c oxidase
Abstract
In cytochrome c oxidase, reduction of molecular oxygen in the active site requires transfer of electrons from cytochrome c towards heme a3. This occurs via a chain of redox intermediates. The dynamics of the final step in this chain, reduction of heme a3 by the chemically identical heme a, cannot be resolved in direct external electron injection experiments. Instead electron redistribution can be monitored upon photodissociation of CO from the mixed-valence (MV) enzyme-CO complex. The published value for the equilibration time in the mitochondrial enzyme is f3 As [1]. However, a detailed spectral analysis of the microsecond data (by Wikstro¨m's group) predicted a substantial additional equilibration phase on a much faster time scale [2]. This prediction was contested (by Brzezinski's group) on the basis of nanosecond experiments [3]. We have performed transient absorption experiments with femtosecond resolution and a time window up to 4 ns under selective excitation of heme a3 in the alpha band (595 nm) starting from the fully reduced (a2+a32+-CO) and MV (a3+a32+-CO) COcomplexes. In the MV complex only, a significant spectral evolution with a nanosecond time constant was observed both in alpha and in Soret spectral regions that can be fully ascribed to electron equilibration (a3+a32+a2+a33+). We suggest that the intrinsic time constant of reduction of heme a3 by heme a is on this time scale. The result will be discussed in the framework of conflicting theoretical predictions. An additional remarkable observation is that the CO-dissociation spectrum from heme a3 depends on the redox state of heme a. This finding adds to the notion that can in principle not be treated as spectrally independent entities. This observation may relate to the strong variation in spectral decompositions using various methods.