The Structure of the Cytochrome P450cam-Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

TitleThe Structure of the Cytochrome P450cam-Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography
Publication TypeJournal Article
Year of Publication2013
AuthorsHiruma, Y., M.A.S. Hass, Y. Kikui, W.M. Liu, B. Olmez, S.P. Skinner, A. Blok, A. Kloosterman, H. Koteishi, F. Lohr, H. Schwalbe, M. Nojiri, M. Ubbink
JournalJournal of Molecular Biology
Volume425
Issue22
Pagination4353-4365
Date PublishedNov 15
ISBN Number0022-2836
Accession NumberWOS:000328100500026
Keywordsbiological electron-transfer, crystal-structures, electron transfer, lanthanide, p450(cam), p450cam monooxygenase, protein complex, protein probe, pseudocontact shift, pseudomonas-putida, putidaredoxin, reduced putidaredoxin, relaxation, system, xplor-nih
Abstract

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 angstrom was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 angstrom, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

DOI10.1016/J.Jmb.2013.07.006
Alternate JournalJ Mol Biol

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