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Pulsed EPR/ENDOR Characterization of Perturbations of the Cu_A Center Ground State by Axial Methionine Ligand Mutations

Slutter, Claire E. and Gromov, Igor and Epel, Boris and Pecht, Israel and Richards, John H. and Goldfarb, Daniella (2001) Pulsed EPR/ENDOR Characterization of Perturbations of the Cu_A Center Ground State by Axial Methionine Ligand Mutations. Journal of the American Chemical Society, 123 (22). pp. 5325-5336. ISSN 0002-7863. https://resolver.caltech.edu/CaltechAUTHORS:20150428-150114010

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Abstract

The effect of axial ligand mutation on the Cu_A site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba_3 has been investigated. The weak methionine ligand was replaced by glutamate and glutamine which are stronger ligands. Two constructs, M160T0 and M160T9, that differ in the length of the peptide were prepared. M160T0 is the original soluble fragment construct of cytochrome ba_3 that encodes 135 amino acids of subunit II, omitting the transmembrane helix that anchors the domain in the membrane. In M160T9 nine C-terminal amino acids are missing, including one histidine. The latter has been used to reduce the amount of a secondary T2 copper which is most probably coordinated to a surface histidine in M160T0. The changes in the spin density in the Cu_A site, as manifested by the hyperfine couplings of the weakly and strongly coupled nitrogens, and of the cysteine β-protons, were followed using a combination of advanced EPR techniques. X-band (∼9 GHz) electron-spin−echo envelope modulation (ESEEM) and two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy were employed to measure the weakly coupled ^(14)N nuclei, and X- and W-band (95 GHz) pulsed electron−nuclear double resonance (ENDOR) spectroscopy for probing the strongly coupled ^(14)N nuclei and the β-protons. The high field measurements were extremely useful as they allowed us to resolve the T2 and Cu_A signals in the g⊥ region and gave ^1H ENDOR spectra free of overlapping ^(14)N signals. The effects of the M160Q and M160E mutations were:  (i) increase in A∥(^(63,65)Cu), (ii) larger hyperfine coupling of the weakly coupled backbone nitrogen of C153, (iii) reduction in the isotropic hyperfine interaction, a_(iso), of some of the β-protons making them more similar, (iv) the a_(iso) value of one of the remote nitrogens of the histidine residues is decreased, thus distinguishing the two histidines, and finally, (v) the symmetry of the g-tensor remained axial. These effects were associated with an increase in the Cu−Cu distance and subtle changes in the geometry of the Cu_2S_2 core which are consistent with the electronic structural model of Gamelin et al. (Gamelin, D. R.; Randall, D. W.; Hay, M. T.; Houser, R. P.; Mulder, T. C.; Canters, G. W.; de Vries, S.; Tolman, W. B.; Lu, Y.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 5246−5263).


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http://dx.doi.org/10.1021/ja003924v DOIArticle
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Additional Information:© 2001 American Chemical Society. Received November 10, 2000. Revised Manuscript Received February 5, 2001. This work has been supported by the German-Israel Foundation for Scientific Research (D.G. and I.P.), National Institutes of Health Grant GM16424 (J.H.R.), and a Feinberg Postdoctoral Fellowship (C.E.S.). C.E.S is deeply indebted to Dr. Angelo Di Bilio, Professor Mike Hill and Professor Harry Gray for numerous discussions about electron transfer, COX, and Cu_A. We thank Dr. Kieron Brown for making the N_2OR coordinates available for us and Professor Peter M. H. Kroneck for the many preprints and the help in getting the N_2OR coordinates. We are grateful to Dr. Frank Neese for the EPR simulation program.
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Funding AgencyGrant Number
German-Israel Foundation for Scientific ResearchUNSPECIFIED
NIHGM16424
Feinberg Postdoctoral FellowshipUNSPECIFIED
Issue or Number:22
Record Number:CaltechAUTHORS:20150428-150114010
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150428-150114010
Official Citation:Pulsed EPR/ENDOR Characterization of Perturbations of the CuA Center Ground State by Axial Methionine Ligand Mutations Claire E. Slutter, Igor Gromov, Boris Epel, Israel Pecht, John H. Richards, and Daniella Goldfarb Journal of the American Chemical Society 2001 123 (22), 5325-5336 DOI: 10.1021/ja003924v
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57060
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:28 Apr 2015 23:13
Last Modified:03 Oct 2019 08:20

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