Inhibition of membrane-bound methane monooxygenase and ammonia monooxygenase by diphenyliodonium: implications for electron transfer.

Diphenyliodonium (DPI) is known to irreversibly inactivate flavoproteins. We have found that DPI inhibits both membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus and ammonia monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a flavoprotein which we proposed catalyzes reduction of the quinone pool by NADH.

Evidence that a type-2 NADH:quinone oxidoreductase mediates electron transfer to particulate methane monooxygenase in methylococcus capsulatus.

NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2).

Detergent solubilization of membrane-bound methane monooxygenase requires plastoquinol analogs as electron donors.

Quinols can provide reducing equivalents for the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membranes. Furthermore, quinols are effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl analog of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) provide the greatest methane monooxygenase activity in whole cells and membrane suspensions, as well as detergent-solubilized samples.

The nature of the copper ions in the membranes containing the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

It is shown that the particulate methane monooxygenase (pMMO) has an obligate requirement for copper. The MMO activity in the particulate fractions obtained from Methylococcus capsulatus (Bath) cells is found to increase with increasing copper content of the membranes. The enzyme activity from membranes obtained from cells grown at low copper levels can be stimulated further by the addition of Cu(II) ions to the assay medium.

Spectroscopic characterization of heme A reconstituted myoglobin.

The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores.