MMO systems

A number of haloalkanes including dichloromethane, chloroform, 1,1-dichloroethane and 1,2-dichloroethane may be degraded by the soluble MMO system of Methylosinus trichosporium (Oldenhuis et al. 1989). 

The methanotroph Methylosinus trichosporium strain OB3b that produces the soluble MMO system consisting of a 40 kDa NADH oxidoreductase, a 245 kDa hydroxylase, and a 16 kDa protein termed component B has a low substrate specificity (Sullivan et al. 1998). 

As indicated by the negative shifts in the reduction potentials of Hox, protein B can interact with the diiron center in Hmv from both MMO systems (63, 64). Consistent with this interpretation are EPR studies of Hmv from both organisms which indicate that, in the presence of protein B, the EPR signal moves from gav 1.83 to gav 1.75 (48, 66). 

The time-dependent, rapid freeze-quench Mossbauer experiments with M. capsulatus (Bath) (51) indicate that decay of the peroxo species proceeds with the concomitant formation of another intermediate, named compound Q. This intermediate, observed in both the M. tri-chosporium OB3b (69, 70) and M. capsulatus (Bath) (51, 71) MMO systems by Mossbauer and optical spectroscopy, decays faster in the presence of substrates. Such behavior indicates that this intermediate is probably on the kinetic reaction pathway for hydroxylation (51, 70). 

Physical studies of the hydroxylase have established the structural nature of the diiron core in its three oxidation states, Hox, Hmv, and Hred. Although the active site structures of hydroxylase from M. tri-chosporium OB3b and M. capsulatus (Bath) are similar, some important differences are observed for other features of the two MMO systems. The interactions with the other components, protein B and reductase, vary substantially. More structural information is necessary to understand how each of the components affects the others with respect to its physical properties and role in the hydroxylation mechanism and to reconcile the different properties seen in the two MMO systems. The kinetic behavior of intermediates in the hydroxylation reaction cycle and the physical parameters of intermediate Q appear similar. The reaction of Q with substrate, however, varies. The participation of radical intermediates is better established with the M. triehosporium... 

B3b system, although it certainly is not ruled out for the M. capsulatus (Bath) enzyme. In comparison to the cytochrome P-450 system, the hydroxylation mechanism for both MMO systems either has a rebound rate constant which is much larger and/or it takes place by an alternative pathway to classical radical rebound. 

One of the most significant results from the advent of these surface science studies on oxides relevant for the present catalytic applications is the fact that oxides can be multiply terminated and that they are not terminated [154, 180, 186-190] in cuts through the bulk structure. This is not unexpected in general [98,156,179] but it is of great value to know this in attempts to understand the mechanisms that activate oxides for catalysis. These rigorous studies must be differentiated from more empirical studies carried out on termination issue with qualitative methods and without predictive power but with the still invaluable advantage that they can be applied [97,191-193] to complex MMO catalyst systems. Such studies can be used to probe the surface reactivity, to address the issue of segregation of, for example, vanadium out of an MMO system and to compare different qualities of the nominally same material with speculative assumptions about the influence of defects. 

All ehemieal, spectroscopic, and kinetic studies of the MMO systems eondueted thus far are consistent with the formation of specific, high affinity complexes between the components. The points of interactions between MMOH and the other two components were first defined using chemical cross-linking procedures. MMOB is readily eross-linked to the MMOH a-subunit while MMOR cross-links to the p-subunit when a water-soluble carbodiimide is reacted with a stoichiometrie mixture of components in solution (Fox et al., 1991). The endogenous tryptophan fluorescence of MMOH and the eoncentration dependence of the MMO components in... 

The oxidation reduction potentials of MMOR have been measured for two MMO systems by monitoring the changes in the optical and EPR spectra. In MMO Bath the potentials for the FAD/FADH [Fe2S2] / [Fe2S2] and FADH/FADH2 couples were found to be nl50, n220, and... 

The MMO system also has a 38-kDa reductase component (MMOR) which has one FAD and one [2Fe-2S] cluster as prosthetic groups [17, 23, 24]. MMOR is readily reduced by NADH and transfers electrons to MMOH. Crosslinking studies show that it has a specific binding site on the MMOH y -subunit [25]. The third component is a 15-kDa protein without cofactors or metal ions termed component B (MMOB). This protein performs a number of regulatory functions through formation of a complex with the a-subunit of MMOH [5, 25]. Some of these functions will be described below, but the net effect is to greatly accelerate the turnover reaction and to increase the product yield from about 40% to nearly 100% when MMOR is also present [26, 27]. 

The search for intermediates in the MMO systems was greatly facilitated by two facts. First, we found that after reduction by two reducing equivalents, MMOH was competent to carry out catalysis in high yield [17]. Consequently, it could be reasonably assumed that all of the mechanistically relevant structural elements were present on this component and any intermediates associated with MMOH were potentially relevant to catalysis. Second, in the presence of MMOB, the rates of formation of intermediates were observed to both be within the time domain accessible to stopped-flow techniques and to decrease as each new intermediate appeared [15]. This meant that the intermediates formed in high yield and could be detected. The intermediates that have been detected thus far and their rates of interconversion at 4°C are summarized in Figure 19-2. The physical properties of the intermediates are sununarized in Table 19-1. 

The radical clock experiments with MMO from M. capsulatus (Bath) and M. trichosporium OB3b carried out in our laboratory indicate that there may not be a single mechanism operative for these enzyme systems. Instead, the mechanism may depend on factors such as the steric and energetic requirements of the substrate, as well as the temperature employed in the MMO hydroxylation reaction. Differences in the MMO systems from the two different organisms include their optimal hydroxylation temperatures as well as sequence variations in the coupling protein B from the two organisms. ... 

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