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https://hdl.handle.net/2440/75471
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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 1432-1327 |
Statement of Responsibility: | 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 |
DOI: | 10.1007/s00775-009-0604-7 |
Published version: | http://dx.doi.org/10.1007/s00775-009-0604-7 |
Appears in Collections: | Aurora harvest Chemistry publications |
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