Methane monooxygenase ,

Methane monooxygenase (MMO) converts methane to methanol according to Eq. (16.1)  

The enzyme ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deox5nibonucleotides, which is the first rate-limiting step in DNA biosynthesis. On the basis of their cofactor compositions, RNRs may be grouped into four different classes [7]. Class I RNR from E. coli is comprised of two homodi-meric subunits, R1 and R2. The R1 subunit (2 x 86kDa) contains the substrate binding site and redox-active cysteine residues, which are involved in the reduction of the ribonucleotides. The R2 subunit (2 x 43 kDa) contains in its active form (R2act) a stable tyrosyl radical (Y122 ), which is necessary for catalytic activity. This tyrosyl radical is located in close proximity to a //-oxo diferric cluster and is embedded about 10 A away from the protein surface [38, 39]. 

It is known that R2act can be reconstituted in vitro from apo-R2 and ferrous ions in the presence of dioxygen and reductant according to Eq. (16.2)  

The reduction of dioxygen to water requires four electrons, three of which are provided by oxidation of the two ferrous ions to ferric ions and by the oxidation of the tyrosine to the tyrosyl radical. The additional electron required by the reaction can be provided in vitro by the addition of reducing agents (ascorbate, excess ferrous ions). The source of the electron in vivo is still unidentified, although iron has been suggested as the origin for this reducing equivalent [48]. 

The in vitro reconstitution reaction of R2act has been studied in detail using a 

MMO shares certain structural similarities with RNR I but MMO probably does not involve free radicals and so was not included above. Lovell et al. [15] have recently reviewed the current state of DFT calculations on both MMO and RNR. 

Afg245 NH2 Fig. 10 Schematic view of large MMO intermediate Q model 

Following the initial work of Siegbahn [69, 70] and Dunietz [66], Lovell et al. [15] built and geometry optimised an impressive 102 atom model (Fig. 10) of intermediate Q which incorporates a number of second- and third-shell amino acid residues [71]. Their choices were guided by their complementary studies of the protein field electrostatics which suggest those residues likely to have a large energetic effect on the active site structure. 

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In one study, a coarse-grained sand aquifer was injected with methane and oxygen to stimulate the production of methane monooxygenase (MMO) enzyme which is capable of degrading TCE (18). TCE, added at 60—100 )-lg/L, was degraded by 20—30%. Injected concentrations of methane and oxygen were approximately 20 mg/L and 32 mg/L, respectively. 

Rubrerythrin (Rr) was first isolated in 1988 from cellular extracts of D. vulgaris Hildenborough (38), and later also found in D. desulfuri-cans (39). Rr is constituted by two identical subunits of 22 kDa and it was shown that each monomer contains one Rd-like center, Fe(RS)4, and a diiron-oxo center similar to the ones found in methane monooxygenase (MMO) (40, 41) or ribonucleotide reductase (RNR-R2) (42). After aerobic purification, the UV-visible spectrum shows maxima at 492, 365, and 280 nm, and shoulders at 570 and 350 nm. This spectrum is similar to the ones observed for Rd proteins. From a simple subtraction of a typical Rd UV-vis spectrum (normalized to 492 nm) it is possible to show that the remainder of the spectrum (maxima at 365 nm and a shoulder at 460 nm) strongly resembles the spectrum of met-hemerythrin, another diiron-oxo containing protein. 

Fox BG, Bomeman JG, Wackett LP, et al. 1990. Haloalkane oxidation by the soluble methane monooxygenase irom Methylosinus trichosporium OB3b Mechanistic and environmental implications. Biochemistry 29 6419-6427. 

Lipscomb ID (1994) Biochemistry of the soluble methane monooxygenase. Annu Rev Microbiol 48 371-399. 

Nguyen H-HT, AK Shiemka, S J Jacobs, BJ Hales, ME Lidstrom, S 1 Chan (1994) The nature of the copper ions in the membranes containing the particulate methane monooxygenase from Methylococcus capsu-latus (Bath). J Biol Chem 269 14995-15005. 

Sontoh S, JD Semrau (1998) Methane and trichloroethylene degradation by Methylosinus trichosporium OB3b expressing particulate methane monooxygenase. Appl Environ Microbiol 64 1106-1114. 

Zahn JA, AA DiSpirito (1996) Membrane associated methane monooxygenase from Methylococcus capsula-tus (Bath). J Bacterial 178 1018-1029. 

Methane monooxygenase may exist in either soluble (sMMO) or particulate (pMMO) forms. These display different substrate ranges and different rates of transformation rates, and most methanotrophs express only the latter form of the enzyme (Hanson and Hanson 1996). The particulate form of methane monooxygenase contains copper, or both copper... 

Enzymes necessary for the metabolism of a substrate may be induced by growth on structurally unrelated compounds. In the examples used for illustration, monooxygenases play a cardinal role as a result of the versatility of methane monooxygenase, while monooxygenases that may be involved in toluene degradation are discussed in Chapter 3, Part 1 and Chapter 8, Part 1. 

FIGURE 7.1 Examples of the reactions catalyzed by methane monooxygenase. 

Fox BG, Wa Froland, JE Dege, JD Lipscomb (1989) Methane monooxygenase from Methylosinus trichospo-rium OB3b. Purification and properties of a three-component system with a high specific activity from a type II methanotroph. J Biol Chem 264 10023-10033. 

Patel RN, CT Hou, Al Laskin, A Felix (1982) Microbial oxidation of hydrocarbons properties of a soluble methane monooxygenase from a facultative methane-utilizing organism, Methylobacterium sp. strain CRL-26. Appl Environ Microbiol 44 1130-1137. 

Oldenhuis R, RLJM Vink, DB Janssen, B Witholt (1989) Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. Appl Environ... 

Jahng D, TK Wood (1994) Trichloroethylene and chloroform degradation by a recombinant pseudomonad expresssing soluble methane monooxygenase from Methylosinus trichosporium OB3b. Appl Environ Microbiol 60 2473-2482. 

According to the following evidences, a-sites are most probably di-iron complexes similar to the di-iron active sites of methane monooxygenase ... 

STUDIES OF THE SOLUBLE METHANE MONOOXYGENASE PROTEIN SYSTEM STRUCTURE, COMPONENT INTERACTIONS, AND HYDROXYLATION MECHANISM... 

Methanotrophs rely on the enzymatic system methane monooxygenase (MMO) to catalyze the first step in the metabolism of methane, shown in Eq. (1) (1, 14). 

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