Methanotrophic bacteria oxidize methane to methanol in the first step of their metabolic pathway. Two forms of methane monooxygenase (MMO) enzymes catalyze this reaction: soluble MMO (sMMO) and membrane-bound or particulate MMO (pMMO). pMMO is expressed when copper is available, and its active site is believed to contain copper. Whereas sMMO is well characterized, most aspects of pMMO biochemistry remain unknown and somewhat controversial. This review emphasizes advances in the past two to three years related to pMMO and to copper uptake and copper-dependent regulation in methanotrophs. The pMMO metal centers have been characterized spectroscopically, and the first pMMO crystal structure has been determined. Significant effort has been devoted to improving in vitro pMMO activity. Proteins involved in sMMO regulation and additional copper-regulated proteins have been identified, and the Methylococcus capsulatus (Bath) genome has been sequenced. Finally, methanobactin (mb), a small copper chelator proposed to facilitate copper uptake, has been characterized.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains ~2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended Xray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 Å. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.Methanotrophs are eubacteria capable of utilizing methane as their only carbon and energy source. Methanotrophs are divided into several classes on the basis of their cell morphologies, membrane arrangements, and pathways for carbon assimilation. The two most widely studied organisms are the type X methanotroph Methylococcus capsulatus (Bath) and the type II methanotroph Methylosinus trichosporium OB3b (1). The first step of their metabolic pathway is the conversion of methane to methanol by the enzyme methane monooxygenase (MMO), 1 † This work was supported by National Institutes of Health Grants HL13531 (to B.M.H.), DK068139 (to T.L.S.), and GM070473 (to A.C.R.). A.S.H. was the recipient of an NSF graduate research fellowship. ‡ The coordinates of Methylosinus trichosporium OB3b pMMO have been deposited in the Protein Data Bank with accession code 3CHX. *To whom correspondence may be addressed. A.C.R.: tel, 847-467-5301; fax, 847-467-6489; e-mail, amyr@northwestern.edu. T.L.S.: tel, 313-577-5712; fax, 313-577-2765; e-mail, tstemmle@med which exists in both a well-studied, but rarely expressed, soluble iron-containing form (sMMO) (2) and a membrane-bound particulate form (pMMO) (2,3). Although the active site and chemistry of sMMO are well established, the nature of the pMMO catalytic center remains controversial, particularly regarding the number and types of metal i...

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.