M ethane-oxidising bac- teria (methanotrophs) are a unique group of Gram-negative bacteria that grow aerobically using methane as the sole carbon and energy source 1 . Over the past 30 years they have received consider- able attention, as they can be exploited in biotransformations and bioremediation 2 , used in the production of single-cell protein and they also play a key role in the cycling of methane in the natural environment. They are readily isolated from a wide variety of environ- ments including freshwater, sediments, soils, seawater, peat bogs and hot springs. The methanotrophs can be divided into two groups based on several morphological and physio- logical characteristics. Type I methanotrophs such as Methylococcus and Methylomonas are -proteobac- teria that possess bundles of unusual intracytoplas- mic membranes throughout the cell and fix carbon into cell biomass using the ribulose monophosphate cycle. Type II methanotrophs such as Methylosinus and Methylocystis are -proteobacteria that have their membranes arranged around the periphery of the cell and fix carbon at the level of formaldehyde via the serine cycle. Methane is oxidised by methanotrophs to CO 2 via the intermediates methanol, formaldehyde and for- mate (Fig. 1). Approximately 50% of the formalde- hyde produced is assimilated into cell carbon and the remainder is oxidised to CO 2 and lost from the cell 3 . The dissimilatory reactions, converting formaldehyde to CO 2 , generate reducing power for biosynthesis and the initial oxidation step. The first enzyme in the methane oxidation path- way is methane monooxygenase (MMO). There are two distinct types of MMO enzymes: a soluble, cyto- plasmic enzyme complex (sMMO) and a membrane- bound, particulate enzyme system (pMMO). sMMO Until recently, it was thought that sMMO was found only in the genera Methylosinus, Methylocystis and Methylococcus. Although it has subsequently been observed in some Methylomonas 4 and Methylomi- crobium 5 species, not all methanotrophs contain this enzyme. sMMO is only ex- pressed when the copper-to- biomass ratio of the culture is low, that is, under ‘low-cop- per’ growth conditions. There is also evidence that copper can inhibit sMMO activity 6 . sMMO has an extremely broad substrate specificity, co-oxi- dizing a wide range of alkanes, substituted aliphatics and even aromatic compounds, making it an extremely attractive enzyme for biotransformation processes and bioremediation 2 . The most well characterized sMMO enzymes are those from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b (Refs 7–9). sMMO is a non-heme, iron-containing enzyme complex consisting of three components: hy- droxylase, protein B and protein C. The hydroxylase has three subunits, ,  and , of ~60, 45 and 20 kDa, respectively, which are arranged in an  2  2  2 config- uration. The  subunit contains a non-heme bis-- hydroxo-bridged binuclear iron centre at the active site of the enzyme, where methanol is formed from methane and oxygen. The sMMO is therefore a member of the family of non-heme binuclear iron proteins, which includes hemerythrin, ribonucleotide reductase and purple acid phosphatase and, together with several other oxygenases, appears to form a subclass of C-H activating oxygenases (Table 1). The crystal structures of hydroxylase components from M. capsulatus (Bath) and M. trichosporium OB3b have been reported 10,11 . The di-iron centre of the hydroxylase resides below the ‘floor’ of two canyon re- gions formed by its  and  subunits. Like some other multicomponent oxygenases, such as phenol hydrox- ylase and toluene monooxygenase, sMMO contains a small regulatory or coupling protein, protein B, the activity and stability of which appears to be controlled at its amino terminus 12 . The structure of protein B from M. capsulatus (Bath) and M. trichosporium OB3b has been determined by NMR, and has provided insights into the interaction of protein B with the hy- droxylase 13,14 . The third component, protein C, is a 39- kDa NADH-dependent [2Fe,2S] and FAD-containing reductase, which accepts electrons from NADH 2 and transfers them to the di-iron site of the hydroxylase. R EVIEWS TRENDS IN MICROBIOLOGY 221 VOL. 8 NO. 5 MAY 2000 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(00)01739-X Many methanotrophs contain both a soluble and a particulate methane monooxygenase. A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes. When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed. A model for the mechanism of this switch is proposed. J.C. Murrell*, I.R. McDonald and B. Gilbert are in the Dept of Biological Sciences, University of Warwick, Coventry, UK CV4 7AL. *tel: +44 2476 523553, fax: +44 2476 523568, e-mail: cm@dna.bio.warwick.ac.uk Regulation of expression of methane monooxygenases by copper ions J. Colin Murrell, Ian R. McDonald and Bettina Gilbert