We have examined mutant forms of MS to examine the role of the His (759), Asp (757) and Ser (810) residues in the hydrogen bonding network. X-ray edge result (in agreement with EPR data) on the Co(II) form of the MS mutant H759G shows that replacing His (759) with a Gly residue causes the formation of a four-coordinate species (identified by the 1s-4pz transition with no Gly ligation to the Co. This mutant shows five magnitude decrease in the catalytic activity with respect to the wild-type Co(II) (Matthews et al., Biochemistry, submitted). This is supported by our earlier result that a four-coordinate Co(II) corrinoid species is very unstable (Wirt et al., J. Am. Chem. Soc., 1993, 115, 5299 and Scheuring et al, J. Phys. Chem., 1996, 100, 3344). The EXAFS results also support a four-coordinate species with an average Co-Neq distance of 1.89+/-0.01 E similar to other cobalamin Hspecies. The Co(II) form of mutant D757N, in which Asp (757) is Hreplaced by a Glu, indicates similar geometry to that of the wild-type HCo(II). EPR data shows that this mutant is 70% bas-off and its Hactivity is impaired by 50-fold. These results show that His (759) in HMS is essential to the catalytic activity and any change in the Hhydrogen bonding network decreases the activity substantially.

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