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Title: Studies of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)
Author: Jiang, Yan
ISNI:       0000 0001 3590 6134
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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Methane monooxygenase catalyses NAD(P)H2- and O2-dependent oxidation of methane to methanol and this reaction initiates the metabolic pathway that supplies the total carbon and energy needs of methanotrophic bacteria. The soluble methane monooxygenase isolated from Methylococcus capsulatus (Bath) is comprised of three proteins: a hydroxylase (Afr. 250 kDa), a reductase {Mr. 38.6 kDa) and a regulator protein (named protein B, Mr. 16 kDa), all three of which are required for enzymatic activity. The hydroxylase is comprised of three polypeptides (a, 60.6 kDa; P, 45.0 kDa; y, 19.8 kDa) of a2P2Y2 stoichiometry and contains a non-heme binuclear iron active centre responsible for methane hydroxylation and a wide variorty of hydrocarbon oxidations. The reductase transfers electrons from NADH to the hydroxylase, and protein B appears to have several regulatory activity. Characterisation of oxygen donor for soluble methane monooxygenase has shown that oxygen is necessary for sMMO-catalysed oxygenation but the high oxygen concentrations result in a decrease of sMMO activity since toxic oxygen species, superoxide anion or peroxide ion, appeared in the reaction system under high oxygen concentration conditions. Characterisation of alternative donors, NO and N2O, demonstrated that they are not suitable for sMMO. Also, the designed experiments to encourage sMMO to catalyse radical recombination reactions were not successful either using non-natural donors or mimicing the conditions used for chemical oxidative coupling. These studies may indicated that upgrading of methane by the native sMMO complex would be impossible. Hydrogen peroxide can replace protein B, the reductase, oxygen and NADH in activation of the hydroxylase of sMMO from Methylococcus capsulatus (Bath) during catalysis of the oxidation of hydrocarbons. Hydrogen peroxide activation of the hydroxylase occurs at the active site iron atoms. The O atom derived from H2O2 is transferred to the substrates and decomposition of peroxide to O2 does not occur in the reaction. The homolytic cleavage of Fe bound O-O- pathway is favoured in the H202-driven system and hydroxyl radicals may be involved in the reaction cycle. Protein B may not only be in controlling electron flow in the sMMO system, but may also be connected with O2 binding to the active site. Proteolysis of the hydroxylase showed that the hydroxylase could be degraded by chymotrypsin and trypsin, but chymotrypsin could greatly degrads the protein resulting in a loss of protein activity. However, even when using a high concentration of trypsin to cleave the hydroxylase, a high percentage of the catalytic activity was still observed. Proteolysis of the hydroxylase by chymotrypsin or trypsin demonstrated that the iron atoms in the hydroxylase active site played an important role in the oxidation of hydrocarbons and that the certain structure of the hydroxylase protein were all necessary for enzyme activity. Chemical modification of the hydroxylase was also studied and the results showed that the hydroxylase protein thermostability could be raised by using crosslinking reagent, polyoxyethylene bis(imidazolyl carbonyl) and that the the PEG-modified hydroxylase could show activity in several organic solvents when H2O2 was used to provide oxygen and electron equivalents.
Supervisor: Not available Sponsor: British Gas plc
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology