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Type: Journal article
Title: Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris
Author: Bell, S.
Xu, F.
Johnson, E.
Forward, I.
Bartlam, M.
Rao, Z.
Wong, L.
Citation: Journal of Biological Inorganic Chemistry, 2010; 15(3):315-328
Publisher: Springer-Verlag
Issue Date: 2010
ISSN: 0949-8257
Statement of
Stephen G. Bell, Feng Xu, Eachan O. D. Johnson, Ian M. Forward, Mark Bartlam, Zihe Rao, Luet-Lok Wong
Abstract: CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe-2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe-S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)(-1)min(-1) for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)(-1 )min(-1) for Pux. This difference mainly arises from weak CYP199A2-PuxB binding (K (m) 34.3 vs. 0.45 microM for Pux) rather than slow electron transfer (k (cat) 19.1 vs. 37.9 s(-1) for Pux). Comparison of the 2.0-A-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin-P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe-2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.
Keywords: Cytochrome P450; Ferredoxin; Electron transfer; Rhodopseudomonas palustris; Mutagenesis
Rights: © SBIC 2009
RMID: 0020121275
DOI: 10.1007/s00775-009-0604-7
Appears in Collections:Chemistry publications

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