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The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus cupsulutus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulutus (Bath) at a modified gold electrode giving rise to two waves at 4 ( t 10) mV and -386(? 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -2 5 ( t 14) mV and -433(+' 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials.Keywords: methane monooxygenase ; hydroxylase component; direct electrochemistry ; differential-pulse voltammetry ; promoter.Methane monooxygenase is an efficient catalyst for the oxidation of methane by molecular oxygen to give methanol as the sole product.There are two forms of the enzyme, soluble (sMMO) and particulate (pMMO), and the former is the more studied. The soluble enzyme from Methylococcus cupsulutus (Bath) [l] consists of an hydroxylase (250.5 kDa), a reductase (38.5 kDa) and a regulatory component, protein B (15.9 kDa) all of which are required for activity. The hydroxylase is made up of two protomers in an a2& arrangement and the X-ray crystal structure of this component has been solved [2-41. The sMMO from Methylosinus trichosporium OB3b has a very similar composition and has also been well characterised [5]. The active site in sMMO is a diiron center, bridged by an hydroxo group (in the resting enzyme), which resides in the a subunit of the hydroxylase. Reducing equivalents from NADH are transferred to the active site through the Fe2S2 and FAD centers in the reductase. Protein B contains no metal ions or cofactors and the details of its regulatory role are unclear.In the resting enzyme the irons are in the fully oxidised Fe(III)Fe(III) state. There are two other oxidation states readily available to the diiron cluster, namely, the mixed valent Fe(III)Fe(II) and the fully reduced Fe(Il)Fe(ll) states. It is the Fe(II)Fe(II) form of the hydroxylase which reacts with and activates 0, during enzyme turnover. The redox potentials of the diiron centers in M . cupsdatus (Bath) and M. trichosporium OB3b have been measured in three independent studiesCorrespondence ro H. A. 0. Hill, New Chemistry Laboratory, South Parks Road, Oxford OX1 3QT, England Ahhreviatinns. sMMO, the soluble form of methane monooxygenase; pMMO, the particulate form of methane monooxygenase; DPV, differential-pulse voltammetry; SPR, surface plasmon resonance; SCE, saturated calomel electrode. MATERIALS AND METHODSGrowth of the M. cupsulutus (Bath) was as previously described [9]. In the purification of the hydroxylase and protein B components, the following modifications...