A Model for Effector Activity in a Highly Specific Biological Electron Transfer Complex:  The Cytochrome P450_cam−Putidaredoxin Couple^,

Abstract

The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.

Comparison of the Complexes Formed by Cytochrome P450cam with Cytochrome b5 and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

“…Furthermore, some ambiguities concerning perturbations in crowded regions of the spectrum have been resolved, and a number of resonances that we previously reported as unperturbed are now seen to be perturbed upon addition of 338, Ile388 and Gln 289 in the β5 sheet. All of these observations support our previous identification of the CYP101 structural features perturbed by binding of Pdx (22). In particular, the first three turns of the C helix (residues 107-118), previously proposed as a primary interaction site for Pdx (19), are now seen, along with the preceding B' helix, to be among the CYP101 structural features most uniformly affected by Pdx binding.…”

“…1A). The apparent exchange rate of the complex at half-saturation was estimated from line width measurements for resonances in the fast-exchange regime to be ~300 s −1 at 17 °C (22,34). As expected, all resonances in the fast exchange regime show 1 H shift perturbations with Δδ max < 300 s −1 , as determined from the value for Δδ max = δ o -δ max obtained from fits of experimental shifts to Eq.…”

“…Recently, we detected a highbarrier conformational shift in the active site of CYP101 that occurs upon Pdx binding, and by comparing chemical shift changes for the 8-CH 3 (which is at slow exchange) and 9-CH 3 (which is at fast exchange) of bound camphor (34), we estimated an exchange rate over this barrier between 150 s -1 and 300 s -1 at 25 °C. Based on the similarities of the rates obtained from two different observations (line widths (22) and camphor 1 H shifts (34), as well as the proximity of NH resonances undergoing slow/intermediate exchange to the active site, we find it likely that the same event is responsible for both observations. The precise nature of this high-barrier event is currently under investigation in our laboratory.…”

“…Protein concentrations and absorbance ratios for both enzymes were estimated by UV-visible spectroscopy using available extinction coefficients (29,30). Conditions for preparation and purification of isotopically-labeled Pdx have been described previously (22).…”

“…All of the NMR spectra were processed and analyzed using the Topspin software package (Bruker Biospin). Conditions for the titration of CYP-S-CO with Pdx r were described previously (22).…”