Teizo Kitagawa

Mechanism of Dioxygen Reduction by Cytochrome c Oxidase as Studied by Time-Resolved Resonance Raman Spectroscopy

Time-resolved resonance Raman (TR3) spectroscopy has been applied to cytochrome c oxidase (CcO) to elucidate the mechanism of dioxygen reduction. Six oxygen isotope-sensitive Raman bands have been identified in the TR3 spectra. The “607-nm species” defined by difference absorption spectrum, which is referenced against the oxidized enzyme, is demonstrated to have an Fe=O heme, although it has long been believed to have an Fe-O-O-X (X=H or CuB) heme. The one-electron reduction of this Fe=O intermediate, which yields the oxoferryl intermediate, is demonstrated to be coupled with proton transfer in the protein. The mechanism of dioxygen reduction by CcO is discussed on the basis of the structures of the reaction intermediates.

Catalytic Roles of the Distal Site Hydrogen Bond Network of Peroxidases

There are highly conserved hydrogen bonds between the distal histidine and adjacent asparagine in many peroxidases. To investigate the functional roles of the hydrogen bond between the distal histidine and Asn-70, Asn-70 in horseradish peroxidase (HRP) was replaced with Val or Asp. The disruption of the Asn-70-His-52 couple decreases the rate of compound I formation to less than 10% of that of native enzyme. Based on resonance Raman spectroscopy, the midpoint pH value of the Fe(II)-His stretching frequency in the acid-base transition was decreased by the mutation of Asn-70, suggesting that the distal histidine became less basic. With a less basic distal histidine, proton abstraction from hydrogen peroxide is harder for the mutants, resulting in remarkable deceleration of compound I formation.