Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase.

We report a protein conformational change following carbon monoxide photodetachment from fully reduced bovine cytochrome c oxidase that is hypothesized to be associated with changes in ligand mobility through a dioxygen access channel in the protein. Although not resolved by earlier photoacoustic or optical studies on this adduct, utilization of slightly lower temperatures revealed a process with a kinetic lifetime of about 70 ns at 10 degrees C. We measure an enthalpy change of about 8 kcal/mol in 0.050 M HEPES buffer that becomes less endothermic (DeltaH approximately 2 kcal/mol) at higher ionic strength. The volume contraction of about -0.7 mL/mol associated with the process almost doubles in higher ionic strength buffer systems. Measurements of samples in phosphate buffer systems are similar and appear to display the same subtle ionic strength dependence. Both the isolation of this photoacoustic signal component and the possible dependence on ionic strength of the thermodynamic parameters derived from its analysis appear analogous to and consistent with prior photoacoustic results monitoring CO photodetachment from the camphor complex of cytochrome P-450. Accordingly, we consider a similar model in which a conformational change results in movement of an exposed charged group or groups towards the interior of the protein, out of contact with solvent, as in the closing of a salt bridge.

Cytochrome rC552, formed during expression of the truncated, Thermus thermophilus cytochrome c552 gene in the cytoplasm of Escherichia coli, reacts spontaneously to form protein-bound 2-formyl-4-vinyl (Spirographis) heme.

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome c(552) in the cytoplasm of Escherichia coli yields both dimeric (rC(557)) and monomeric (rC(552)) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC(557) is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276, 6537-6544]. Unexpectedly, the monomeric rC(552) transforms spontaneously to a cytochrome-like chromophore having, in its reduced state, the Q(oo) transition (alpha-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC(552), at 1.41 A resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH(2)-S-CH(2)-C(alpha)] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 A resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q(oo)() transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm(-1) for oxidized and at 1646 cm(-1) for reduced samples, and the greatly altered, paramagnetically shifted (1)H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c.