MMOH

Physical Properties of MMOH from M. trichosporium OB3b and M. capsulatus (Bath)... 

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]. 

The best characterized of the BMMs are the sMMOs (Figure 13.24), which are the only members of the family capable of activating the inert C-H bond of methane, one of the most difficult reactions in nature to achieve. Like most members of the BMM superfamily, sMMO requires three protein components, the hydroxylase MMOH, which contains the carboxylate-bridged diiron centre, a regulatory protein MMOB and a [2Fe-2S]- and FAD-containing reductase (MMOR) which shuttles electrons from NADH to the diiron centre. 

Figure 13.24 Structures of sMMO components and proposed reaction cycle, (a) MMOH (b) the MMOR FAD and ferredoxin (Fd) domains (c) MMOB. In MMOH the ot, P and y subunits are coloured blue, green and purple, respectively. Iron, sulfur and FAD are coloured orange, yellow and red, respectively and are depicted as spheres. The MMO reaction cycle is shown on the right, with atoms coloured [Fe (black), C (grey), O (red) and N (blue)]. (Reprinted with permission from Sazinsky and Lippard, 2006. Copyright (2006) American Chemical Society.)...

The hydroxylase component (MMOH) is composed of two a(3y protomers which use protein contacts between each of the a and (3 subunits along a two-fold symmetry axis to form an ot2[S2Y2 heterodimer. The interface between the two protomers forms a large cleft within which the MMOR is thought to dock. The diiron centre is located within a four-helix bundle made up of helices B, C, E and F of the a subunit. Helices E and F are on the surface of the hydroxylase, forming part of the rim of the cleft with the diiron centre some 12 A beneath this rim. Figure 13.25 compares the four-helix bundles that contain the diiron centres in six representatives of the superfamily. 

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. 

Proteins with dinuclear iron centres comprise some well studied representatives like ribonucleotide reductase (RNR), purple acid phosphatase (PAP), methane monooxygenase hydroxylase (MMOH), ruberythrin and hemerythrin. The latter is an oxygen carrier in some sea worms it has been first characterized within this group and has thus laid the foundation to this class of iron coordination motif. Ruberythrin is found in anaerobic sulfate-reducing bacteria. Its name implies that, in addition to a hemerythrin-related diiron site another iron is coordinated in a mononuclear fashion relating to rubredoxin which is an iron-... 

Figure 2.4 X-ray crystal structures of different forms of MMOH from M. capsulatus (Bath) coordinates taken from PDB files 1FYZ (a) [59] and 1FZ6 (b) [60]. Coordinative bonds are represented bysolid linesand hydrogen bondsor weak bonds by dotted lines. Only the amino acid side-chains of the active site and coordinated aqua/hydroxido/methoxido ligands are shown. Hydrogen atoms have been omitted for clarity.

Scheme 2.7 The catalytic cycle of MMOH based on experimental and computational results [41]. The amino acids are numbered according to the sequence of M. capsulatus (Bath).

Whereas several transient species have been observed for dioxygen activation by MMOH, no intermediates were found by rapid-mixing spectroscopic methods for the actual methane hydroxylation step. Mechanistic probes, i.e. certain non-natural substrates that are transformed into rearranged products only if the reaction proceeds via a specific intermediate such as a radical or a cation, give ambivalent results Some studies show that products according to a pathway via cationic intermediates are obtained in sMMO hydroxylations and at least one study suggests the presence of a radical intermediate [40]. Computational analyses of the reaction of MMOHq with methane suggest a so-called radical recoil/rebound mechanism in which MMOHq... 

An additional pathway of similar energy was found to proceed via a concerted oxygen atom insertion [45]. In summary, it appears as if different substrates can be differently activated and the reaction pathway hence proceeds via different intermediates. Clearly, more studies are necessary before the mechanism of methane hydroxylation by MMOH can be fully understood. 

MMOH-P converts in a first-order process to Q, the species directly respon-... 

---