220 Biochimica et Biophysica Acta 952 (1988) 220-229 Elsevier BBA 33036 Spectroscopic properties of the hydroxylase of methane monooxygenase Roger C. Prince, Graham N. George, Judith C. Savas, Stephen P. Cramer * and Ramesh N. Patel * * Exxon Research and Engineering Co., Annandale, NJ (U.S.A.) (Received 27 July 1987) Key words: Methane monooxygenase; Hydroxylase; Spectroscopic property; ESR; X-ray spectroscopy The hydroxylase component of methane monooxygenase (EC 1.14.13.25), which catalyzes the oxidation of methane to methanol, has been studied by visible, electron spin resonance and X-ray spectroscopies. The enzyme appears to possess a/~-oxo- or bt-hydroxo-bridged binuclear iron site, with no sulfur ligands to the cluster. Each Fe has 4-6 oxygen (or nitrogen) ligands, at an average distance of 1.92 + 0.03 A. The Fe-Fe distance is 3.05 + 0.05 A. Essentially all of the irons are in the Fe 3+ state as the enzyme is prepared, but reduction with N-methylphenazonium methosulfate generates ESR-detectable states that appear to emanate from mixed-valence binuclear sites. One of these, with gay near 1.85, displays typical Curie law microwave saturation behavior, but the other, with ga, near 1.73, has a very potent method of spin-relaxation. Together they account for approximately 0.6 spins per molecule. Introduction The diverse group of organisms capable of liv- ing on methane as the sole source of carbon and energy were first described at the beginning of the century. Nevertheless, it has only been in the last decade or so that their physiology, ecology and biochemistry have received much attention (see Refs. 1 and 2). Methanotrophic bacteria can be divided into two classes, I and II, dependent on the structure of their internal membranes and on the pathways of assimilation of one carbon units; Type I organisms use the ribulose monophosphate * Present address: Schlumberger-Doll Research, Ridgefield CT 06877-4108, U.S.A. * * Present address: E.R. Squibb and Sons, P.O. Box 191, New Brunswick, NJ 08903, U.S.A. Correspondence: R.C. Prince, Exxon Research and Engineer- ing Co., Clinton Township, Route 22 East, Annandale, NJ 08801, U.S.A. pathway for the assimilation of carbon at the level of formaldehyde, and have bundles of internal membranes throughout the cell that appear to be invaginations of the cytoplasmic membrane. Type II organisms assimilate formaldehyde by the serine pathway, and have paired peripheral membranes within the cell [1]. Both types of organisms metabolize methane by first hydroxylating it to methanol, using the enzyme methane mono- oxygenase (EC 1.14.13.25). Under some condi- tions this activity is found in soluble extracts of cells; under others, the activity appears to be associated with the membrane. There is some con- fusion over what regulates the apparently different activities, but high copper concentrations or low oxygen tension favor the membrane-bound en- zyme [3,4]. Both soluble [5,6] and membrane-bound [7] forms of the enzyme system have been isolated, but the former has been rather more char- acterized. Dalton and his colleagues have isolated a three-component system from Methylococcus capsulatus (Bath) composed of a hydroxylase 0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)