RESOLUTION OF THE ELECTRONIC-TRANSITIONS OF CYTOCHROME-C-OXIDASE - EVIDENCE FOR 2 CONFORMATIONAL STATES OF FERROUS CYTOCHROME-A

Second-derivative absorption spectra are reported for a variety of oxidation and ligation states of bovine cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1). The high resolving power of the second-derivative method allows us to assign the individual electronic transitions of cytochrome a and cytochrome a3 in many of these states. In the fully reduced enzyme, one observes a single electronic transition at 444 nm, corresponding to the Soret transition for both ferrous cytochrome a and ferrous cytochrome a3. When the cytochrome a3 site is occupied by an exogenous ligand (CN or CO), one observes two absorption bands assignable to the ferrous cytochrome a chromophore, one at ca. 443 nm and the other at ca. 450 nm. The appearance of the 450-nm band is dependent only on ligand occupancy at the cytochrome a3 site and not on the oxidation state of the cytochrome a3 iron. These results can be interpreted either in terms of a heterogeneous mixture of two ferrous cytochrome a conformers in the cytochrome a3-ligated enzyme or in terms of a reduction in the effective molecular symmetry of the ferrous cytochrome a site that results in a lifting of the degeneracy of the lowest unoccupied molecular orbital associated with the Soret-pi,pi* transition of cytochrome a. In either case, the present data indicate that ferrous cytochrome a can adopt two distinct conformations. One possible structural difference between these two states could be related to differences in the strength of hydrogen bonding between the ferrous cytochrome a formyl oxygen and a proton donor from an unidentified amino acid side chain of the enzyme. The implications of such modulation of hydrogen-bond strength are discussed in terms of possible mechanisms of proton translocation and electron transfer in the enzyme.