Resonance Raman studies of lactoperoxidase, its catalytic intermediates and model compounds
Abstract
Lactoperoxidase is a mammalian enzyme that is part of our natural defense system. Very little is known about this enzyme, and so studies have been designed to gain an understanding of how this enzyme works in nature. Enzymes are responsible for catalyzing biochemical reactions. Often times the enzyme's shape changes during its enzymatic cycle. Resonance Raman (RR) spectroscopy is capable of monitoring the structural changes occurring during this process. By examining the enzyme at various points in time one can develop a picture of how the enzyme changes as the biochemical reaction proceeds. All attempts to generate and monitor the enzymatic intermediates of lactoperoxidase have been unsuccessful. Currently, little is being done on RR studies of these intermediates owing to their inherent photo-lability, fast reaction times and large-scale quantities of sample required for these experiments. Recent advances made in the area of time-resolved resonance Raman spectroscopy provide an opportunity to structurally characterize the enzymatic intermediates of lactoperoxidase. The other area of interest centers on studying the factors, which dictate the reactivity of the prosthetic group, i.e., heme chromophore, of the lactoperoxidase enzyme. Recently, a method for extracting the heme prosthetic group of lactoperoxidase, without modification, has been published. The inherent problem with removing the heme prosthetic group of lactoperoxidase is that the surrounding apoprotein matrix is covalently linked to the heme through the 1 and 5 methyl substituents. These covalent linkages, which occur only for the mammalian peroxidases, have hindered its removal from the protein, preventing systematic studies of its inherent reactivity. The generation of the intermediates and the extraction of the heme prosthetic group have been accomplished. The first intermediates appear to be a ferryl heme π-cation radical that subsequently decays into a second intermediate that is considered to be a ferryl protein-centered radical. The heme prosthetic group for lactoperoxidase, heme 1, has been successfully extracted without modification, and from these studies it has been proposed that the LPO(N), resting state enzyme, is a six coordinate high-spin complex.
This paper has been withdrawn.