Implication of the redox behavior of the copper cluster in the particulate methane monooxygenase on the methane hydroxylation mechanism
Date Issued
2005
Date
2005
Author(s)
Wang, Vincent Cho-Chien
DOI
en-US
Abstract
Potentiometric titrations of the copper centers in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) are described. The reduction potentials of the various copper centers were measured by poising the enzyme in pMMO-enriched membranes in the presence of different redox mediators in an electrochemical cell, and quickly freezing the sample to record their low temperature EPR signals. Measurements of the low temperature EPR intensities of the type 2 sites and trinuclear Cu(II) centers yielded [Cu(I)]/[Cu(II)] for the various copper centers at the various different potentials, and the mid-point potentials were determined from the Nernst equation. The measurements were performed on two different forms of the enzyme in pMMO-enriched membranes: (i) the enzyme as isolated aerobically, but in the absence of hydrocarbon substrate; and (ii) the same preparation oxidized under air in the presence of acetylene or other hydrocarbon substrates. While the E-cluster copper sites exhibited essentially the same high reduction potential of +490 mV, substantially different results were obtained for the redox behavior of the C-cluster copper ions between the two forms. The reduction potentials of the oxidized C-cluster copper sites observed in the EPR were found to be substantially lower compared to that of the E-clusters. Accordingly, the redox potential of the oxidized bis(μ–oxo)dicopper(III) and bis(μ–peroxo)dicopper (II) dimer associated with the isolated type 2 site of the same cluster must be considerably higher than +490 mV, and there must be a kinetic barrier for transfer of electrons from the E-clusters to this site. This “splitting” of the redox potential of the trinuclear copper cluster is artifactual of the non-physiological state of enzyme created from the oxidation of the six copper ions by eight oxidizing equivalents from two dioxygen molecules. When the enzyme is turning over in the presence of hydrocarbon substrate, the dioxygen chemistry is tightly linked to the hydroxylation chemistry to achieve kinetic competence, and the redox potentials of the C-cluster copper ions were found to be substantially higher.
The results of the present study add a significant chapter to our understanding of the structure and function of pMMO. First, the potentiometric titrations confirmed without any further question the existence of C-cluster and E-cluster copper ions, the number of distinct C-clusters, as well as the functional role they play in the methane hydroxylation chemistry, as the various copper centers are distinguished by distinct redox behaviors in the EPR, as manifested by their different appearance in the EPR spectrum at different potentials. Second, the different redox behavior of the C-clusters copper ions between the “as-isolated” enzyme and during turnover in the presence of hydrocarbon substrate has led to important insights into the details of how electron transport, dioxygen chemistry and hydrocarbon oxidation are linked in this complex system.
Subjects
微粒體甲烷單氧化酵素
電子自旋光譜
pMMO
monooxygenase
potentiometric titration
EPR
Type
thesis
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