Abstract
Hemoglobin and myoglobin are known to undergo autoxidation, in which the oxyferrous form of the heme is oxidized to the ferric state by O2. Dehaloperoxidase-A (DHP-A), a multifunctional catalytic hemoglobin from Amphitrite ornata is an exception and is observed to undergo the reverse process, during which the ferric heme is spontaneously reduced to the oxyferrous form under aerobic conditions. The high reduction potential of DHP (+202mV at pH 7.0) partially explains this unusual behavior, but the endogenous source of reducing equivalents has remained obscure. Cysteine, methionine, tyrosine, and tryptophan are the principal endogenous reducing agents in proteins that may explain the observed autoreduction in DHP-A. In fact, DHP-A has six methionines, which may be of particular importance for the observed autoreduction. To investigate the role of the sulfur-containing residues, we created seven mutants (C73S, C73S/M49C, S78C, M63L, M64L, M63L/M64L, and H55V) by site-directed mutagenesis and conducted a series of CO-driven autoreduction kinetic measurements. Mutational analysis suggests a role for the pair of methionines M63 and M64 increaing the autoreduction rate. Adding surface cysteines has little effect, but the C73S mutation that eliminates the only native surface cysteine accelerates the autoreduction process. The kinetics had a sigmoidal form which was found to be a result of anti-cooperative behavior. This observation suggests that DHP-A's monomer-dimer equilibrium in solution may play a role in regulating the autoreduction process.
Published Version
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