LAUE DIFFRACTION STUDY ON THE STRUCTURE OF CYTOCHROME-C PEROXIDASE COMPOUND-I

Background: Cytochrome c peroxidase from yeast is a soluble haem-conta ining protein found in the mitochondrial electron transport chain wher e it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient i ntermediate (termed compound I) in the reaction of cytochrome c peroxi dase with hydrogen peroxide. This intermediate contains a semistable f ree radical on Trp191, and an oxyferryl haem group. Results: Compound I was produced in crystals of yeast cytochrome c peroxidase by reactin g the crystalline enzyme with hydrogen peroxide in a flow cell. The re action was monitored by microspectrophotometry and Laue crystallograph y in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels f or an additional half an hour. The structure of the intermediate was d etermined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processe d with a new software package (LEAP), overcoming many of the former pr oblems encountered in extracting structural information from Laue expo sures. Conclusions: The geometry of the haem environment in this prote in allows structural changes to be extremely small, similar in magnitu de to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electro ns with a minimal need for structural adjustment.