Massimo Sassaroli

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.

Structure and Dynamics of Photodissociated Myoglobin

The molecular basis of ligand binding in heme proteins and the mechanisms which control ligand affinity are essential to determine for a complete understanding of the biological function of this ubiquitous class of proteins. An approach to this understanding is to determine the properties of the ligand-free photoproducts which may be generated by photodissociation of the carbon monoxide bound adducts (1, 2). If the photoproducts can be studied under conditions in which the protein has not fully relaxed to its deoxy conformation, then a metastable species along the pathway from the ligand-bound protein to the deoxy protein will be available for examination. By utilizing resonance Raman scattering, changes in the biological center of this molecule, the heme, may be followed.