| The methanotrophic bacterium, Methylococcus capsulatus, is capable of the conversion of methane to methanol at ambient temperature and pressure. In this manuscript, we show that quinols can provide reducing equivalents to the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membrane fractions. Additionally, quinols are shown to be effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl-analog of plastoquinol and duroquinol provide the greatest MMO activity in whole cells and membrane fractions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytically-active methane monooxygenase. This detergent was also utilized when reincorporating the endogenous quinone, 18'-methylene CoQ8, during analysis of pMMO enzymatic activity supported by endogenous quinones. The endogenous quinone was capable of supporting activity of pMMO in detergent-solubilized fractions, when used in combination with NADH. However, the synthetically reduced form of the endogenous quinone is incapable of supporting activity in whole cells, membranes, or the solubilized fraction. Finally, oxygen uptake studies of whole cells, membranes, and solubilized membranes in addition to UV-visible spectroscopic studies reveal the existence of an alternative oxidase in vitro. The inhibitors octyl gallate and cyanide inhibit oxygen uptake in both membranes and whole cells. |
