Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3b(T) or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO, activities in the 290 nmol propylene oxidized min(-1) (mg protein)(-1) range, activities approaching 400 nmol propylene oxidized min(-1) (mg protein)(-1) were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity. The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0-5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of < 0-4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in < 2 min. At Cu(II)-to-mb ratios between 0.4 and 0.9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or cluroquinol) and substrates (CH4 and/or O-2). The results indicated that Cu-mb increased electron flow to the pMMC, increased the free radical formed following the addition Of 02 and decreased the residual free radical following the addition Of O-2 Plus CH4. The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.