A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy.

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.

The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris.

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.

Crystal structure of a ferredoxin reductase for the CYP199A2 system from Rhodopseudomonas palustris.

Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k(cat) = 37.9 s(-1) with 4-methoxybenzoic acid). The reduction of Pux by PuR shows K(m) = 4.2 microM and k(cat) = 262 s(-1) in 50 mM Tris, pH 7.4. K(m) is increased to 154 microM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 A resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site.

Tailoring an alien ferredoxin to support native-like P450 monooxygenase activity.

A ferredoxin associated with biological Fe-S cluster assembly has been remodelled to transfer electrons to a P450 enzyme and support substrate oxidation at 80% of the physiological ferredoxin activity, opening up the possibility of tailoring ferredoxins to reconstitute the activity of P450 enzymes for which the electron transfer partner proteins are not known.

The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site.

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.

Selective oxidative demethylation of veratric acid to vanillic acid by CYP199A4 from Rhodopseudomonas palustris HaA2.

CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).