Redox behavior of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The redox properties of the copper in particulate methane monooxygenase fro m Methylosinus trichosyorium OB3b were investigated. The ESR spectrum of th e pMMO-containing membranes from M. trichosyorium OB3b indicated a typical type II copper (II) signal (g(parallel to) = 2.24, A(parallel to) = 18.4 mT , g(perpendicular to) = 2,06, alpha(2) = 0.84). By anaerobic addition of ex cess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra i ndicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the cop per is the active site of pMMO or an electron carrier. During the titration , the intensity of the type II copper signal decreased with decreasing pote ntial and the multiple hyperfine structure at g = 2.06 appeared clearly. Al though the copper signal did not change by treatment of the EDTA-treated me mbranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest th at some redox mediators play a role as an electron carrier between the acti ve site and a reductant, and the presence of at least two types of copper s ites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E- site).