The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity

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The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the formyl hemis were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemis was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the α-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings. © 1984.

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Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular

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