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10116 Biochemistry 1993 32 10116 10124 Dynamics of Protein Relaxation in Site Specific Mutants of Human Myoglobin David G Lambright Sriram Balasubramanian and Steven G Boxer Department of Chemistry Stanford University Stanford California 94305 5080 Received February 22 1993 Revised Manuscript Received June 29 1993 ABSTRACT We have recently reported spectroscopic evidence for structural relaxation of myoglobin Mb following photodissociation of MbCO Lambright D G Balasubramanian S Boxer S G 1991 Chem Phys 158 249 2601 In this paper we report measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket positions 45 64 and 68 in order to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding The kinetics of ligand binding and conformational relaxation were monitored by transient absorption spectroscopy in the Soret spectral region and the results are analyzed using a four state ligand binding model Two principal results emerge 1 amino acid substitutions in the distal heme pocket affect the kinetics of the nonequilibrium conformational relaxation and 2 the rate of ligand escape from the protein matrix is not significantly perturbed by the distal heme pocket mutations Much of our knowledge regarding the mechanism of ligand binding to myoglobin Mb comes from extensive studies of CO rebinding to photolyzed Mb in viscous glycero1 water solutions see for example Austin et al 1975 Beece et al 1980 Hasinoff 1981 Ansari et al 1986 and Campbell et al 1987 j In glycerol glasses below 200 K the kinetics of CO rebinding to Mb are highly nonexponential and this has been interpreted as reflecting a distribution of barrier heights for the iron ligand bond formation step process I Austin et al 1975 A variety of theoretical models have been developed to explain this basic observation Agmon Hopfield 1983

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