Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris
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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)−1min−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 μM for Pux) rather than slow electron transfer (k cat 19.1 vs. 37.9 s−1 for Pux). Comparison of the 2.0-Å-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.
KeywordsCytochrome P450 Ferredoxin Electron transfer Rhodopseudomonas palustris Mutagenesis
The authors would like to thank the reviewers for helpful suggestions that helped improve the manuscript. This work was supported by the Higher Education Funding Council for England, the Engineering and Physical Sciences Research Council, and the Biotechnology and Biological Sciences Research Council (EP-D048559-1), UK, and the National Science Foundation of China (30221003).
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