Dioxygen activation and substrate hydroxylation by the hydroxylase component of toluene/O-xylene monooxygenase from pseudomonas sporium OX1

Non-heme carboxylate-bridged diiron centers in the hydroxylase components of the bacterial multicomponent monooxygenases activate dioxygen at structurally homologous active sites. Catalysis requires the management of four substrates: electrons, protons, dioxygen, and hydrocarbons. Protein component complexes control the delivery of these substrates to the diiron center in the hydroxylase ensuring selective hydrocarbon oxidation. A detailed mechanistic understanding of structural and chemical consequences of such interactions is a significant challenge. This thesis begins with an overview of our current understanding of these processes. The discussion is primarily on the methane monooxygenase systems (MMO) because these have been the most extensively studied BMMs to date. Recent results for the toluene/o-xylene monooxygenase (ToMO) and phenol hydroxylase systems from Pseudomonas sporium OX1 are also briefly summarized, the former being the research focus of this dissertation. Restricting access to the diiron center in ToMOH and other non-heme carboxylate-bridged diiron proteins was proposed to facilitate observation of oxygenated intermediates. To examine this hypothesis, dioxygen activation in ToMOH mutants that were predicted to occlude this channel was investigated by rapid-freeze quench (RFQ) EPR, Mossbauer, and ENDOR spectroscopy and stoppedflow optical spectroscopy. For the I100W mutant, a transient species is observed with an absorption maximum at 500 nm. EPR and Mossbauer spectra of RFQ samples identified this species as a diiron(III,IV) cluster spin-coupled to a neutral W radical. ENDOR spectra of this intermediate confirmed the protonation state and type of the amino acid radical and also identified a labile terminal water or hydroxide on the diiron center.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Murray, Leslie Justin

Advisor dc:contributor.advisor

• Stephen J. Lippard.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/41556

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/41556