TIME RESOLUTION OF STRUCTURE CHANGES IN METALLOENZYMES

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The nature and function of metal atoms in enzymes and proteins has been a keystone of biomedical research since Warburg's initial discoveries in the 1930's on "heavy-metal enzyme catalysis" and Keilin's identification of hemoproteins. Many biophysical approaches to the study of these heavy metal enzymes have been in process. Optical and electron paramagnetic resonance studies are good examples of those that give definitive information on many properties but have "blind spots" that fail to give incisive information on the metal atoms in all valences or states, i.e., certain properties are "invisible" to most techniques. K edge absorption of X-rays by heavy metal atoms occurs in all valence states and gives definitive fingerprints of the nature of such atoms and their electronic configuration and chemical environment. Extended edge absorption studies (exafs) give many other properties, some of which can be interpreted as precise distance measurements to neighbors of the heavy metal atoms. While related data may ultmately be inferred from x-ray or nuclear magnetic resonance data, the great potential advantages of the exafs method are the possibilities of structural studies of the metalloproteins that are time resolved at enzymatically functional concentraions. Optical and epr sample monitoring detects any change of valence or liganding under synchrotron irradiation. This proposal seeks to set up instrumentation development and testing of components that increase the efficiency of photon collection from x-irradiated samples, the ultimate goal being to achieve edge absorption EXAFS and anomalous scattering studies in the range appropriate enzyme activity studies, i.e., less than 100 microns M and eventually extending down toward micromolar concentrations. Concomitant with this development are essential innovations necessary for kinetic studies of the state of mental atoms in enzymatic function. Last but not least are improved methods for on-line sample monitoring to ensure the integrity of the biological material throughout the course of the x-irradiation.