Manganese peroxidase (MnP), which normally oxidizes Mn2+ to Mn3+, is rapidly and completely inactivated in an H2O2-dependent reaction by 2 equivalents of sodium azide. The inactivation is paralleled by formation of the azidyl radical and high yield conversion of the prosthetic heme into a meso-azido adduct. The meso-azido enzyme is oxidized by H2O2 to a Compound II-like species with the Soret band red-shifted 2 nm relative to that of native Compound II. The time-dependent decrease in this Compound II-like spectrum (t( 1/2 ) = 2.3 h) indicates that the δ-meso azido heme is more rapidly degraded by H2O2 than the prosthetic heme of control enzyme (t( 1/2 ) = 4.8 h). MnP is also inactivated by phenyl-, methyl-, and ethylhydrazine. The phenylhydrazine reaction is too rapid for kinetic analysis, but K(I) = 402 μM and k(inact) = 0.22/min for the slower inactivation by methylhydrazine. Reaction with phenylhydrazine at pH 4.5 does not yield iron-phenyl, N-phenyl, or meso-phenyl heme adducts. Ethylhydrazine inactivates the enzyme both at pH 4.5 and 7.0, but only detectably produces δ-meso-ethyl-heme at pH 7.0. Reconstitution of apo-MnP with hemin or δ-meso-ethylheme yields enzyme with, respectively, 50 and 5% of the native activity. The δ-meso-alkyl group thus suppresses most of the catalytic activity of the enzyme even though a Compound II-like species is still formed with H2O2. Finally, Co2+ inhibits the enzyme competitively with respect to Mn2+ but does not inhibit its inactivation by azide or the alkylhydrazines. The results argue that substrates interact with the heme edge in the vicinity of the δ-meso-carbon. They also suggest that Mn2+ and Co2+ bind to a common site close to the δ-meso-carbon without blocking the approach of small molecules to the heme edge. An active site model is proposed that accommodates these results.
|ジャーナル||Journal of Biological Chemistry|
|出版ステータス||出版済み - 1991|
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