The pH dependence of native PHM and its M314H variant have been studied in detail. For WT PHM the intensity of the Cu-S interaction visible in the Cu(I) EXAFS data is inversely proportional to catalytic activity over the pH range 3 - 8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive met-on form and an active flexible met-off form which derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to an evolution of this model where the met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% due to effects on both kcat and KM but it displayed the same activity/pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra which could be simulated by replacing the CuM Cu-S(Met) interaction with a Cu-N/O but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5 the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the met-on form observed at low pH in WT cannot be due to a strengthening of the CuM-methionine interaction, but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers which brings a new S-donor residue into a favorable orientation for coordination to copper and generating an inactive form. Cys coordination is unlikely since all Cys residues in PHM are engaged in disulfide crosslinks. Sequence comparison with the PHM homologues TBM and DBM suggest that M109 (adjacent to the H-site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 protonates with a pKA of 4.6 to generate the inactive low-pH form with CuH coordinated by M109, H107 and H172.
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