Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidas e (EPO), and lactoperoxidase (LPO), homologous members of the mammalian per oxidase superfamily, can all serve as catalysts for generating nitric oxide -(nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prost hetic groups that are ligated through a histidine nitrogen and use H2O2 as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic an d/or conformational changes in their heme pockets using a combination of ra pid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe( III) state] causes immediate buildup of the corresponding Fe(III) complexes . Spectral changes indicate that two LPO-Fe(II) species are present in solu tion at equilibrium. Analyses of stopped-flow traces collected when EPO, MP O, or LPO solutions rapidly mixed with NO were accurately fit by single-exp onential functions. Plots of the apparent rate constants as a function of N O concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed sig nificantly lower affinity toward NO compared to Fe(II) forms, suggesting th at heme reduction causes a dramatic change in the heme pocket electronic en vironment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-s pin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward bindin g of diatomic ligands. Our results suggest that, upon reduction, the heme p ocket of MPO collapses, LPO adopts two spectroscopically and kinetically di stinguishable forms (one partially open and the other relatively closed), a nd EPO remains open.