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NMR Studies of Cytochrome P-450_(scc). Effects of Steroid Binding on Water Proton Access to the Active Site of the Ferric Enzyme

Jacobs, Russell E. and Singh, Jangbir and Vickery, Larry E. (1987) NMR Studies of Cytochrome P-450_(scc). Effects of Steroid Binding on Water Proton Access to the Active Site of the Ferric Enzyme. Biochemistry, 26 (14). pp. 4541-4545. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20160516-132218706

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Abstract

Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450(P-450_(scc)) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe^(3+) center, and the long electron spin relaxation time of the heme iron, T_s~0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is ~2.5 Å, a distance consistent with a water molecule binding directly to the heme iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to ~4 Å, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the Complex with (22R)-22-hydroxycholesterol, a distance of ~2.7 Å is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450_(scc) with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3β-ol, or (20R)-20-phenyl-5- pregnene-3β,20-diol, also yielded distances of ~2.5 Å and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand. In contrast to cytochrome P-450_(cam), in which water is excluded from the heme center in the substrate complex [Griffin, B. W., & Peterson, J. A. (1975) J. Biol. Chem. 250, 6445-6451; Philson, S. B., Debrunner, P. G., Schmidt, P. G., & Gunsalus, I. C. (1979) J. Biol. Chem. 254, 10173-10179], protons have rapid access to regions near the active site of several steroid complexes of P-450_(scc). This suggests that the active site of P-450_(scc) may be open to solvent and that solvent water molecules, rather than acid/base groups in the active site, may provide the protons required during the monooxygenation reaction cycle.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi00388a056DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi00388a056PublisherArticle
ORCID:
AuthorORCID
Jacobs, Russell E.0000-0002-1382-8486
Additional Information:© 1987 American Chemical Society. Received October 28, 1986; Revised Manuscript Received March 13, 1987. This work was supported by National Institutes of Health Grant AM30109 and National Science Foundation Grant PCM-8309976. L.E.V. is the recipient of Research Career Development Award AM01005 from the National Institutes of Health. The 500-MHz NMR experiments were performed at the Southern California Regional NMR Center (California Institute of Technology) supported by NSF Grant CHE79-16324.
Funders:
Funding AgencyGrant Number
NIHAM30109
NSFPCM-8309976
NIHAM01005
NSFCHE79-16324
Issue or Number:14
Record Number:CaltechAUTHORS:20160516-132218706
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160516-132218706
Official Citation:NMR studies of cytochrome P-450scc. Effects of steroid binding on water proton access to the active site of the ferric enzyme Russell E. Jacobs, Jangbir Singh, and Larry E. Vickery Biochemistry 1987 26 (14), 4541-4545 DOI: 10.1021/bi00388a056
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67135
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:16 May 2016 21:42
Last Modified:03 Oct 2019 10:03

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