474 Abstracts

151 C O N F O R M A T I O N A L M U L T I P L I C I T Y A N D D Y N A M I C S OF C Y T O C H R O M E P450 - T H E CO S T R E T C H M O D E A S S P E C T R O S C O P I C P R O B E

C. Jung, H. Sehulze, J. Contzen and O. Ristau Max-Delbrzick-Centrum for Molecular Medicine, Robert-ROssle-Strasse 10, D-13122 Berlin, Germany

The catalytic function of cytochrome P450 results from the complex interaction of coordination chemical properties of the heme and the structural and dynamic behavior of the protein. Many spectroscopic methods have been used to characterize the heme pocket structure in hemoproteins. The stretch mode of the iron bound CO ligand measured by infrared spectroscopy have been found as a powerful tool to analyze the active site in diverse hemoglobins [ 1 ]. We demonstrate the application of this tool for characterization of the active site in cytochrome P450. Systematic studies of the effect of solvent conditions, temperature, pressure and substrate binding on the CO stretch infrared spectrum [2-4] revealed two phenomena: (i) CO stretch frequency shifts and (ii) infrared band broadening and CO stretch mode multiplicity. Correlations of the CO stretch frequency at room temperature with the high- spin content, initially induced by the different substrates in the oxidized state of P450, with the P450 redox potential, and with 13C chemical shift of the bound CO ligand [5] let suppose that the polarity in the heme pocket determines the frequency. There is a relation between the frequency of the bound CO (A state) and of the photodissociated CO (B state) at 20K which let suggest that the bound CO ligand has contact to a polar group of the surrounding protein. A CO stretch mode multiplicity is observed for substrate-free P450 which can be interpreted as conformational substates of the protein. The substate equilibrium is strongly dependent on temperature, pressure, and solvent. Substrate binding drastically changes this equilibrium. The temperature dependence of the width of the infrared band representing a conformational substate will be discussed in terms of inhomogeneous and homogeneous broadening effects [2]. The structural properties of the active site of cytochrome P450 extracted from the infrared studies may help to understand functional properties. Regarding the CO complex as model for the physiologically relevant dioxygen complex we suggest that the switch of the P450 catalysis toward the uncoupling pathway, induced by specific substrates , is connected with a loosening of a polar contact between the bound ligand and the protein.

I. G.B. Ray, X.-X. Li, J.A. Ibers, J.L. Sessler and T.G. Spiro, J. Am. Chem. Soc. 116, 162 (1994)

2. C. Jung and F. Marlow, Studia Biophysica Acta 120, 241 (1987) 3. C. Jung, G. Hui Bon Hoa, K.-L. SchrOder, M. Simon and J.P. Doucet, Biochemistry

31, 12855 (1992) 4. H. Schuize, O. Ristau and C. Jung, Eur. J. Biochem. 224, 107 (1994) 5. N. Legrand, A. Bondon, G. Simonneaux, C. Jung and E. Gill, FEBS Letters 1995,

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