Role of tyrosine-103 in myoglobin peroxidase activity: kinetic and steady-state studies on the reaction of wild-type and variant recombinant human myoglobins with H(2)O(2).

Myoglobin (Mb) catalyzes a range of oxidation reactions in the presence of hydrogen peroxide (H(2)O(2)) through a peroxidase-like cycle. C110A and Y103F variants of human Mb have been constructed to assess the effects of removing electron-rich oxidizable amino acids from the protein on the peroxidase activity of Mb: a point mutation at W14 failed to yield a viable protein. Point mutations at C110 and Y103 did not result in significant changes to structural elements of the heme pocket, as judged by low-temperature electron paramagnetic spectroscopy (EPR) studies on the ground-state ferric proteins. However, compared to the native protein, the yield of globin radical (globin*) was significantly decreased for the Y103F but not the C110A variant Mb upon reaction of the respective proteins with H(2)O(2). In contrast with our expectation that inhibiting pathways of intramolecular electron transfer may lead to enhanced Mb peroxidase activity, mutation of Y103 marginally decreased the rate constant for reaction of Mb with H(2)O(2) (1.4-fold) as judged by stopped-flow kinetic analyses. Consistent with this decrease in rate constant, steady-state analyses of Y103F Mb-derived thioanisole sulfoxidation indicated decreased V(max) and increased K(m) relative to the wild-type control. Additionally, thioanisole sulfoxidation proceeded with lower stereoselectivity, suggesting that Y103 plays a significant role in substrate binding and orientation in the heme pocket of Mb. Together, these results show that electron transfer within the globin portion of the protein is an important modulator of its stability and catalytic activity. Furthermore, the hydrogen-bonding network involving the residues that line the heme pocket of Mb is crucial to both efficient peroxidase activity and stereospecificity.