Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase

Myeloperoxidase is the most abundant, protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central ro le in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidi zed by myeloperoxidase, and we have determined the features that enable the m to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxid ase, The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s(-1) (15 degrees C, pH 7.0) and were relatively insensitive to the substit uents on the aromatic ring. Rate constants for reduction of compound II var ied between 6.5 x 10(5) M-1 s(-1) for ABAH and 1,3 x 10(3) M-1 s(-1) for 4- nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compo und I and compound II by BAHs adhered to the Hammett rule, and there were s ignificant correlations with Brown-Okamoto substituent constants. This indi cates that the rates of these reactions were simply determines by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydro gen peroxide because it underwent auto-oxidation, Although BAHs generally r eacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxid ase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constant s. BAHs containing more hydrophilic substituents were more effective at con verting the enzyme to compound III, This implies that BAH radicals must hyd rogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate co nstants for reduction of compounds I or II. We propose that BAHs destroy th e heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidas e-hydrogen peroxide complex.