Formate dehydrogenase (NAD+ depet) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isobted anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 celluloe and Sephacryl S-300 colns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the a-subunit was 53,800 ± 2,800, and that of the a-subunit was 102,600 ± 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent a2j2 arrangemnt. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Among the large number of microorganisms that have formate dehydrogenase, the NAD-linked enzyme (EC 1.2.1.2) has been isolated from both facultative methylotrophic bacteria (3, 13) and several yeast species (20; also reference 4 and references therein). There is a great deal of heterogeneity in the structure and composition of bacterial formate dehydrogenases. They range from molecules with two identical subunits and no prosthetic group, found in facultative methylotrophic strains of Moraxella (30) In methylotrophs, formate dehydrogenase is a critical component involved in the oxidative metabolism of methane and methanol. The NADH generated by this enzyme is, in some cases, believed to be the cell's only source of reductant (2). In Methylosinus trichosporium, NAD-linked formate dehydrogenase functions as an in vitro electron donor to methane monooxygenase (32) and indirectly to nitrogenase via a ferredoxin-NAD+ reductase and ferredoxin (9,10) (Fig. 1). Although formate dehydrogenase plays an important role in the metabolism of nonfacultative methylotrophs (and obligate methanotrophs), this enzyme has not yet been isolated from either of these groups of bacteria. In this study we purified the formate dehydrogenase from the obligate methanotroph M. trichosporium and report on its structural and regulatory characteristics. * Corresponding author. MATERIALS AND METHODSGrowth conditions. M. trichosporium OB3b cells used for formate dehydrogenase preparations were batch cultured in 9-liter carboys on nitrate-containing medium (11) as described previously (10). In these cultures, the air-methane mixture (5:1) was dispersed by passing it through stone diffusers and stirring the culture slowly with a magnetic bar. Our cultures did not attain a high density (80 to 120 Klett units, no. 66 filter) in these batch cultures and were harvested by centrifugation after 6 days of growth. After harvesting, the cells were immediately frozen (unwashed)

A gene, fdxP, was identified upstream of the rrnA gene in Caulobacter crescentus and shown to encode ferredoxin II (FdII) by insertional inactivation. FdII is homologous to a class of [2Fe-2S] ferredoxins typified by putidaredoxin. Furthermore, reconstitution assays demonstrated that FdII was able to promote p-hydroxybenzoate hydroxylase activity in ferredoxin-depleted extracts. Thus, biodegradation of p-hydroxybenzoate may be ferredoxin dependent in C. crescentus.