Methane monooxygenase, MMO

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. 

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). 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

Methane monooxygenase (MMO) Methanogenic bacteria Methane —> methanol... 

A new class of metalloprotelns containing polynuclear, non-heme oxo-bridged iron complexes has emerged recently. Dinuclear centers are present in hemerythrin (Hr), ribonucleotide reductase (RR), purple acid phosphatases (PAP) and, possibly, methane monooxygenase (MMO) these centers as well as model compounds are reviewed in Chapter 8. 

The multiprotein complex methane monooxygenase (MMO) serves meth-anotrophs to convert methane to methanol. It can be either soluble (sMMO) or membrane bound ( particulate , pMMO) and it typically consists of three components, a reductase (MMOR), a component termed protein B (MMOB) and a hydroxylase denoted MMOH. The nature of the metal cofactors in the latter component are reasonably well understood for sMMO as will be discussed in the non-heme iron section. For the pMMO of Methylococcus capsulatus an obligate requirement for copper was shown. As reported in reference 1 a trinuclear Cu(II) cluster was discussed128 but the number and coordination of coppers still is a matter of continuing investigation since then. 

Since H202 is easier to handle than 02, we will focus on the use of the former. Many metals can be used for this transformation [50]. Among them, iron compounds are of interest as mimics of naturally occurring non-heme catalysts such as methane monooxygenase (MMO) [51a] or the non-heme anti-tumor drug bleomycin [51b]. Epoxidation catalysts should meet several requirements in order to be suitable for this transformation [50]. Most importantly they must activate the oxidant without formation of radicals as this would lead to Fenton-type chemistry and catalyst decomposition. Instead, heterolytic cleavage of the 0—0 bond is desired. In some cases, alkene oxidation furnishes not only epoxides but also diols. The latter transformation will be the topic of the following section. 

Figure 1. Comparison of the coordination spheres of the dinuclear iron sites in oxidized hemerythrin (Hr), ribonucleotide reductase (RNR), uteroferrin (Uf), and methane monooxygenase (MMO).

Cardy, D. L., Laidler, V., Salmond, G. P, and Murrell, J. C., 1991b, Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b, Mol. Microbiol. 5 335n342. 

---