Conformational Change Induced by Putidaredoxin Binding to Ferrous CO-ligated Cytochrome P450cam Characterized by 2D IR Spectroscopy.

Sashary Ramos,Edward J Basom,Megan C Thielges

doi:10.3389/fmolb.2018.00094

Sashary Ramos, Edward J Basom + Show 1 more

Open Access

https://doi.org/10.3389/fmolb.2018.00094

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Abstract

The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O2 complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx.

Highlights

IntroductionThe population of and interconversion among multiple conformations and substates of a hierarchical energy landscape
Protein function requires dynamics, the population of and interconversion among multiple conformations and substates of a hierarchical energy landscape
The reduced, CO-bound camphor complex of wt P450cam in the absence and presence of Pdx and of L358P first were characterized by linear spectroscopy (Figure 2)

Summary

Introduction

The population of and interconversion among multiple conformations and substates of a hierarchical energy landscape They have been implicated in the activity of cytochrome P450s (CYPs), a superfamily of heme-thiolate enzymes that catalyze oxygen insertion from molecular dioxygen in diverse substrates for a wide range of biosynthetic and metabolic processes (Sigel et al, 2007; Ortiz de Montellano, 2015). The binding of substrate to the ferric enzyme displaces a heme-ligated water molecule, leading to conversion of the heme from low to high spin and an increase in redox potential. This promotes a one-electron reduction, followed by binding of O2. P450cam is an outlier as the second reduction step has a strict requirement for the native redox partner, the iron-sulfur protein putidaredoxin (Pdx) (Figure 1; Tyson et al, 1972)

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