Hot‐Spot Residues in the Cytochrome P450cam–Putidaredoxin Binding Interface

Abstract

Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.

Identification of productive and futile encounters in an electron transfer protein complex

“…However, the crystal and solution structures of the ET complex formed by the two molecules were solved only very recently by X-ray crystallography and paramagnetic NMR (23, 24). The relative orientation of the proteins within the complex in the solution and crystalline states is practically identical, consistent with the information available from the mutagenesis studies of the system and favorable for effective ET (21,[25][26][27][28][29]. It is still under debate whether Pdx induces opening of substrate-bound cytP450cam.…”

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula

“…Thus, waste of electrons by PdR and PdX, which generates reactive oxygen species, is negligible when electrons flow to CYP101A1 in any PUPPETs during the monooxygenation reaction. The apparent specific activity of PUPPET M1M2M3 at 40 nM protein concentration (1.1 × 10 3 min −1 ) was almost half of the maximum turnover number of CYP101A1 (2.4 × 10 3 min −1 ), which is reported to be obtained in the presence of 1 µM PdR and 100 µM PdX, and 2.1‐fold higher than that of PUPPET S1S2S3 , which is identical to a previously reported PUPPET …”

“…This weak association requires micromolar concentrations of ferredoxins for sufficient electron the donation to P450s, which precludes practical in vitro applications . For example, putidaredoxin (PdX), which is a redox partner of a regio‐ and stereo‐specific d ‐camphor hydroxylating P450 (CYP101A1) from Pseudomonas putida , binds to CYP101A1 with a dissociation constant of 2 µM and to putidaredoxin reductase (PdR) with a dissociation constant of 37 µM . Consequently, CYP101A1 generally requires more than 10 µM PdX to achieve maximum catalytic activity (≈2.4 × 10 3 min −1 ) …”

Spectroscopic studies of the cytochrome P450 reaction mechanisms

“…Several new residues were found at the interface and their importance for CYP101A1-Pdx interactions was confirmed in kinetic measurements. [409] Another recent NMR study further explored the multiple configurations of encounter complexes of Pdx and CYP101A1 and located the preferred configurations at the most favorable spots on the map of electrostatic potential. [401]…”