INFRARED STUDY OF OXIDASE DYNAMICS

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Time-resolved infrared (TRIR) studies of protein dynamics on all timescales from subpicosecond to seconds are proposed. The primary targets of this study are cytochrome oxidases from mammals and bacteria. Some work on myoglobin and hemoglobin is suggested to establish technical feasibility or to model the more complicated oxidases. The long-term goal is to understand in structural detail the functional dynamics of the oxidases, upon which all aerobic life on earth depends. A technical objective is to develop new and generally applicable approaches to the study of protein dynamics, based upon the observation by infrared of structural features that are invisible to other spectroscopic probes. A suite of TRIR techniques will be used: optical delay methods for subpicosecond to nanosecond dynamics; real-time TRIR for dynamics from nanoseconds to seconds; time-resolved infrared linear dichroism for direct determination of the relative orientation of infrared-active structures and electronic dipoles, and step-scan time-resolved FAIR for observation of transient phenomena over the entire infrared spectrum. These studies will be supplemented where appropriate by transient electronic spectroscopy, time- resolved magnetic circular dichroism, and time-resolved resonance Raman investigations. Specific studies to be performed include: (1) TRIR investigation of ligation dynamics, including photodissociation/recombination of heme-bond ligands, fate of ligands after photodissociation, binding to nonheme metals, and dependence of dynamics upon steric and electronic factors. The result will be detailed understanding of ligation and steric phenomena at the site of O2 activation. (2) TRIR studies of endogenous protein IR chromophores, including amino acid side chains, the peptide backbone and prosthetic groups. The results will elucidate the response of the protein structure and conformation to ligation and redox perturbations. (3) Time-resolved infrared linear dichroism, studied in connection with (1) and (2) above, aimed at determining specific structural reorientations which occur during the short-timescale dynamics.