A novel multistep mechanism for oxygen binding to ferrous hemoproteins: rapid kinetic analysis of ferrous-dioxy myeloperoxidase (compound III) formation.

Myeloperoxidase (MPO), a hemoprotein that uses H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions, is implicated as a participant in inflammatory injury and cardiovascular diseases. Mechanisms for turning off this enzyme once released, preventing unwanted tissue injury, are poorly understood. We recently demonstrated that MPO heme reduction causes collapse of the heme pocket, as monitored by significant reductions in the rates of diatomic ligand binding to the heme iron. Using spectral and rapid kinetic measurements, we now demonstrate that molecular oxygen (O(2)) binds to ferrous MPO (MPO-Fe(II)) in a distinct and novel mechanism. Rather than occurring through a simple, reversible, one-step mechanism, as is typical for O(2) binding to other ferrous hemoproteins, the reaction involves several kinetically and spectroscopically distinguishable intermediates. Diode array spectrophotometric and stopped-flow studies reveal that the formation of the MPO-Fe(II)-O(2) complex consists of at least three elementary steps and includes at least two sequential transient intermediates. The first step involves reversible formation of a transient intermediate via an O(2)-dependent mechanism, followed by two sequential O(2)-independent steps that appear to be conformational in origin. Insights into mechanisms for inactivating MPO and the novel mode of O(2) binding to the hemoprotein may provide important clues toward understanding the catalytic action of MPO.