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PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function

Lu, Yu-Jhang and Hung, Mu-Cheng and Chang, Brian T.-A. and Lee, Tsu-Lin and Lin, Zhi-Han and Tsai, I-Kuen and Chen, Yao-Sheng and Chang, Chin-Shuo and Tsai, Yi-Fang and Chen, Kelvin H.-C. and Chan, Sunney I. and Yu, Steve S.-F. (2019) PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function. Journal of Inorganic Biochemistry, 196 . Art. No. 110691. ISSN 0162-0134. https://resolver.caltech.edu/CaltechAUTHORS:20190417-075957631

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Abstract

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu^I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB_(33–414) and PmoB_(55–414), each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM Cu^(II), it behaves like M. capsulatus (Bath) cultured under high copper stresswith abundant membrane accumulation and high CuI content. The recombinantPmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-rayabsorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu^I proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB_(33–414) mutant. Reaction of the dicopper site with dioxygenproduces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.jinorgbio.2019.04.005DOIArticle
ORCID:
AuthorORCID
Chan, Sunney I.0000-0002-5348-2723
Yu, Steve S.-F.0000-0002-3462-065X
Alternate Title:PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function
Additional Information:© 2019 Published by Elsevier Inc. Received 2 September 2018, Revised 28 February 2019, Accepted 8 April 2019, Available online 15 April 2019. This work was supported in whole or in part by Academia Sinica (AS-107-TP-ML07 and AS-KPQ-106-DDPP) and grants from the Ministry of Science and Technology, Taiwan (MOST 94-2113-M-001-016, 101-2113-M-001-007-MY3 and 104-2113-M-001-013-MY3 to S.S.F.Y.; and MOST 101-2113-M-001-013 to S.I.C.). We are grateful to Dr. Jyh-Fu Lee and his staffs at the National Synchrotron Radiation Research Center, Hsinchu, Taiwan, for their kind assistance with the X-ray absorption measurements. Mr. Tai-Lang Lin assisted us with the electron microscopy analyses at the EM Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica. The contributions of the coauthors to this article are: Y.-J.L., Z.-H.L., T.-L.L. and Y.-S.C. constructed and designed the expression vectors for protein expression; M.-C.H., C.-S.C. and Y.-S.C. conducted the mutagenesis studies; Y.-J.L., M.-C.H., B.T.-A.C. and C.-S.C. carried out the specific activity measurements on the recombinant PmoB constructs; B.T.-A.C. purified the MBP-PmoB55–414; Y.-J.L., B.T.-A.C., Y.-F.T. and S.S.-F.Y. conducted the EPR and/or XAS experiments; M.-C.H., B.T.-A.C., Z.-H.L., I.-K.T., C.-S.C. and Y.-S.C. performed the quantification of the copper contents in the proteins, and the protein quantification using antibodies; B.T.-A.C., Z.-H. L., I.-K.T. and C.-S.C. grew the E. coli cells for the biochemical and biophysical characterization of the recombinant proteins; K.H.-C.C., S.S.-F.Y. and S.I.C. designed the research; S.S.-F.Y. and S.I.C. wrote the paper.
Funders:
Funding AgencyGrant Number
Academia SinicaAS-107-TP-ML07
Academia SinicaAS-KPQ-106-DDPP
Ministry of Science and Technology (Taipei)94-2113-M-001-016
Ministry of Science and Technology (Taipei)101-2113-M-001-007-MY3
Ministry of Science and Technology (Taipei)104-2113-M-001-013-MY3
Ministry of Science and Technology (Taipei)101-2113-M-001-013
Subject Keywords:Copper sites; Particulate methane monooxygenase (pMMO); Membrane protein; Methane oxidation; Oxygen/hydrogen-peroxide redox loop; X-ray absorption spectroscopy
Record Number:CaltechAUTHORS:20190417-075957631
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190417-075957631
Official Citation:Yu-Jhang Lu, Mu-Cheng Hung, Brian T.-A. Chang, Tsu-Lin Lee, Zhi-Han Lin, I-Kuen Tsai, Yao-Sheng Chen, Chin-Shuo Chang, Yi-Fang Tsai, Kelvin H.-C. Chen, Sunney I. Chan, Steve S.-F. Yu, The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function, Journal of Inorganic Biochemistry, Volume 196, 2019, 110691, ISSN 0162-0134, https://doi.org/10.1016/j.jinorgbio.2019.04.005. (http://www.sciencedirect.com/science/article/pii/S0162013418305166)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94736
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Apr 2019 22:03
Last Modified:03 Oct 2019 21:06

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