Yuan-Chin Ching

Proton translocation in cytochrome c oxidase : redox linkage through proximal ligand exchange on cytochrome a3

An analysis of resonance Raman scattering data from CO-bound cytochromec oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochromea3 in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochromea3 can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochromec oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochromea3 (Shapleighet al., 1992; Hosleret al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O2-binding site based on proximal ligand exchange between tyrosine and histidine on cytochromea3. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.

The Role of Water Near Cytochrome a in Cytochrome c Oxidase

Resonance Raman scattering studies of cytochrome c oxidase reveal that two vibrational modes narrow upon placing the enzyme in D2O. This is interpreted as evidence for the presence of water molecules near cytochrome a that increase the linewidth of the heme modes due to resonance vibrational energy transfer to the H2O bending mode. From the nature of the modes in which the broadening is detected, it is deduced that the water molecules are located near the formyl and the vinyl substituents of the cytochrome a. The change in width in the formyl mode appears quickly, whereas that in the vinyl mode only develops after extended exposure of the enzyme to D2O. On the basis of these results we propose a new mechanism for proton translocation. In this hypothesis water molecules at the active site become activated and are dissociated into protons and hydroxyl groups due to changes in the pKas of residues near the heme when the redox state of the cytochrome a changes. Structural features of the protein stabilize this charge separation and allow directional migration of protons to the cytosolic side of the inner mitochondrial membrane. It is pointed out that this mechanism may be operative in all proton-translocation complexes, and it is observed that in bacteriorhodopsin, also a proton pump, water molecules are detected near the active site lending support to the generality of this mechanism.

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.

Proton translocation in cytochrome c oxidase: Redox linkage through proximal ligand exchange on cytochrome a3

An analysis of resonance Raman scattering data from CO-bound cytochromec oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochromea3 in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochromea3 can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochromec oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochromea3 (Shapleighet al., 1992; Hosleret al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O2-binding site based on proximal ligand exchange between tyrosine and histidine on cytochromea3. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.

Intermediates Formed in the Reaction of Cytochrome c Oxidase with Oxygen

Cytochrome c oxidase is the terminal enzyme in the electron transport chain. In this role, it catalyzes the transfer of four electrons from cytochrome c to di-oxygen, the physiological substrate. Oxygen binds to cytochrome c oxidase on the microsecond time scale and is fully reduced to water in milliseconds. An understanding of the molecular basis for the catalytic mechanism of the oxygen reduction has been sought after for many years but has remained elusive due to the difficulty in establishing the identity of each intermediate on this time scale by optical spectroscopic techniques. Recently, we [1–5] and others [6–11] have applied resonance Raman spectroscopy to the study of the oxygen reduction process and achieved considerable success. In this paper, we discuss the findings from the resonance Raman scattering experiments and based on these results present a model for the catalytic pathway.