The redox metalloproteins are necessary for the life processes of all organisms. Fundamental studies of the active sites of redox metalloproteins, including systems ranging in complexity from relatively simple electron-transfer proteins to """"""""multiheaded"""""""" oxidases, are proposed. The objectives are to understand, in terms of the fundamental physical and chemical properties of the metal sites and their interactions with the protein structure, how these proteins accomplish their functions of facile electron transfer and the activation of small molecules such as oxygen. Such understanding is crucial in view of the large number of pathological conditions which are related to the function of the redox metalloproteins. The primary experimental approach proposed is vibrational spectroscopy, including resonance Raman (RR) and Fourier transform infrared (FTIR). Emphasis is placed upon: RR and FTIR studies as a function of temperature between cryogenic and ambient conditions; and time-resolved vibrational spectroscopy (resonance Raman TR3, FTIR, TR-FTIR) of transients in the ligation and electron transfer dynamics. The initial target systems are the simple copper """"""""blue"""""""" proteins, e.g., plastocyanin, azurin, and stallacyanin; the multicopper oxidases, e.g. the laccases, ascorbate oxidase, and ceruloplasmin; and cytochrome oxidase. All of these systems will be studied by cryo-vibrational techniques. Expected results include: improved spectral resolution and signal-to-noise, leading to more reliable analysis of the vibrational data in the conventional sense; from the temperature dependence of frequencies and line-shapes, information on fundamental vibrational dynamics and their possible influence on the electron transfer process; and possible observation of very low energy electronic transitions such as the spin-spin interactions in the type 3 copper pair or in cytochrome a3 of cytochrome oxidase. The TR3 and TR-FTIR studies are directed at the multi-copper oxidases and cytochrome oxidase. Expected results include: elucidation of the structures of transients in the function of the enzymes at all timescales; modes of 02 binding before reduction of 02; oxidation and spin states of hemes in cytochrome oxidase transients; and effects of protein constraints on transient structures of the metal sites. Given improved vibrational data from the cryogenic studies, including isotope sites. Given improved vibrational data from the cryogenic studies, including isotope data, application of modern small-molecule vibrational analysis methods to the metalloprotein active sites is proposed. The experimental results and the analysis thereof will lead to reliable structural, dynamical and functional interpretations of vibrational data.
