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Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Spradlin, Jessica and Lee, Diana and Mahadevan, Sruthi and Mahomed, Mavish and Tang, Lawrence and Lam, Quan and Colbert, Alexander and Shafaat, Oliver S. and Goodin, David and Kloos, Marco and Kato, Mallory and Cheruzel, Lionel E. (2016) Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies. Biochimica et Biophysica Acta - Proteins and Proteomics, 1864 (12). pp. 1732-1738. ISSN 1570-9639 . http://resolver.caltech.edu/CaltechAUTHORS:20161003-124138537

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Abstract

Background: In order to perform selective C-H functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)_2PhenA (bpy = 2,2′-bipyridine and PhenA = 5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective C-H bond hydroxylation of several substrates. Methods: A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. Results: The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. Conclusions: In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General significance: The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bbapap.2016.09.005DOIArticle
http://www.sciencedirect.com/science/article/pii/S157096391630187XPublisherArticle
Additional Information:© 2016 Elsevier B.V. Received 3 August 2016; Received in revised form 9 September 2016; Accepted 13 September 2016; Available online 14 September 2016. L.E.C. gratefully acknowledges financial support by the National Institute of Health through grant number SC3 GM095415. J.S. would like to thank the NSF RUMBA program at SJSU. D.G. thanks financial support by the National Institute of Health through grant number GM41049-27. L.E.C. is thankful for the use of facilities at the Beckman Institute Laser Resource Center and to the director Jay R. Winkler for helpful discussion. We thank Prof. Ilme Schlichting for help with the protein crystallization and data collection. Diffraction data were collected on beamline X10SA at the Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland. We thank the PXII staff for their support in setting up the beamline. M.Kl. gratefully acknowledges support and funding by the Max Planck Society.
Funders:
Funding AgencyGrant Number
NIHSC3 GM095415
NSFUNSPECIFIED
NIHGM41049-27
Max Planck SocietyUNSPECIFIED
Subject Keywords:Cytochrome P450; Electron transfer; Hybrid P450 BM3 enzymes; Crystal structure; Enzyme catalysis; Photocatalytic activity
Record Number:CaltechAUTHORS:20161003-124138537
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20161003-124138537
Official Citation:Jessica Spradlin, Diana Lee, Sruthi Mahadevan, Mavish Mahomed, Lawrence Tang, Quan Lam, Alexander Colbert, Oliver S. Shafaat, David Goodin, Marco Kloos, Mallory Kato, Lionel E. Cheruzel, Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1864, Issue 12, December 2016, Pages 1732-1738, ISSN 1570-9639, http://dx.doi.org/10.1016/j.bbapap.2016.09.005. (http://www.sciencedirect.com/science/article/pii/S157096391630187X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:70757
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Oct 2016 21:54
Last Modified:03 Oct 2016 21:54

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