Аннотация
Several molecular mechanisms for cleavage of the oxalate carbon-carbon
bond by manganese-dependent oxalate decarboxylase have recently
been proposed involving high oxidation states of manganese. We have
examined the oxalate decarboxylase from Bacillus subtilis by electron
paramagnetic resonance in perpendicular and parallel polarization
configurations to test for the presence of such species in the resting
state and during enzymatic turnover. Simulation and the position
of the half-field Mn(II) line suggest a nearly octahedral metal
geometry in the resting state. No spectroscopic signature for Mn(III)
or Mn(IV) is seen in parallel mode EPR for samples frozen during
turnover, consistent either with a large zero-field splitting in
the oxidized metal center or undetectable levels of these putative
high-valent intermediates in the steady state. A narrow, featureless
g = 2.0 species was also observed in perpendicular mode in the presence
of substrate, enzyme, and dioxygen. Additional splittings in the
signal envelope became apparent when spectra were taken at higher
temperatures. Isotopic editing resulted in an altered line shape
only when tyrosine residues of the enzyme were specifically deuterated.
Spectral processing confirmed multiple splittings with isotopically
neutral enzyme that collapsed to a single prominent splitting in
the deuterated enzyme. These results are consistent with formation
of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly
enhanced relaxation relative to abiological tyrosyl radicals was
observed, but site-directed mutagenesis indicated that conserved
tyrosine residues in the active site do not host the unpaired spin.
Potential roles for manganese and a peripheral tyrosyl radical during
steady-state turnover are discussed. 10.1074/jbc.M402345200
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