The C-Terminal Aqueous-Exposed Domain of the 45 kDa Subunit of the Particulate Methane Monooxygenase in Methylococcus capsulatus (Bath) Is a Cu(I) Sponge

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• 作者：Steve S.-F. Yu ; Cheng-Zhi Ji ; Ya Ping Wu ; Tsu-Lin Lee ; Chien-Hung Lai ; Su-Ching Lin ; Zong-Lin Yang ; Vincent C.-C. Wang ; Kelvin H.-C. Chen ; Sunney I. Chan
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 4, 2007
• 年：2007
• 卷：46
• 期：48
• 页码：13762 - 13774
• 全文大小：451K
• 年卷期：v.46,no.48(December 4, 2007)
• ISSN：1520-4995

文摘

The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcuscapsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystalstructure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is basedmight be missing many of the important metal cofactors, including the putative trinuclear copper clusterat the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a bufferof reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry[Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., andChan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for atrinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposeddomains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for theE-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposedsubdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178)and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperativelywhen cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition,circular dichroism measurements reveal that the C-terminal subdomain folds into a -sheet structure inthe presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with thehigh redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of theE-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of theenzyme.