Nature of the Energy Landscape for Gated Electron Transfer in a Dynamic Redox Protein

Abstract

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms.

ELDOR Spectroscopy Reveals that Energy Landscapes in Human Methionine Synthase Reductase are Extensively Remodelled Following Ligand and Partner Protein Binding

“…Electron-electron double resonance (ELDOR) can be used to measure the distances between two unpaired electrons, for example, the semiquinone state of flavin cofactors, [19] as we demonstrated recently with human cytochrome P450 reductase (CPR). [7] We now show that ELDOR spectroscopy can also give direct information on the conformational landscape of human MSR in the diradical (FAD and FMN semiquinone) state. Using ELDOR spectroscopy we have identified multiple conformational intermediates in MSR and analysed their relative stabilities.…”

“…[3] By analogy with other diflavin oxidoreductases related to MSR, such as nitric oxide synthase [4] and cytochrome P450 reductase, [5][6][7] it has been suggested that MSR fluctuates between "closed" and "open" conformations. In this simple model, one conformation ("open" conformation) of MSR presents the FMN domain so that it is available for interaction with the AD of MS; in another conformation (a "closed" conformation) the FMN domain is in close proximity to the NADPH/FAD-binding domain, where it facilitates interflavin electron transfer (Figure 1).…”

“…[4,5,8,[12][13][14][15] These mechanisms are supported by computational, spectroscopic and kinetic studies that suggest electron transfer is limited by conformational motion. [7,[16][17][18] Despite their importance, the experimental exploration of energy space across the conformational landscape is challenging. Important questions about the nature of landscapes remain unanswered: for example, are energy landscapes smooth or rugged; are they remodelled following ligand and partner protein binding, or solvent perturbation; what is the extent of the landscape in spatial terms?…”

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

“…Otherwise, the reduced FMN would have too restricted solvent accessibility in the closed conformation to efficiently deliver electrons to the substrates (30)(31)(32). It has been also found that the open conformational state is significantly populated in the NADPH-bound, reduced state of diflavin reductases (31,33,34). At variance with the stable interaction between the FMN-binding domain of Ndor1 and the unstructured region of anamorsin, the transient interaction observed in the regions containing the FMN and [2Fe-2S] cofactors can be easily regulated upon the occurrence of the conformational closed-to-open equilibrium of the FAD/FMN domains of Ndor1.…”

Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c

“…9), whereas resting state CPR exists in a predominant "closed conformation" in which the FMN cofactor is in close proximity with FAD but buried in the interface between the FMN and FAD binding domains, making it inaccessible by cyt c (7,52). A movement of the FMN binding domain relative to the rest of the CPR molecule is essential for electron transfer from CPR to its redox partners, the process of which may be regulated by nucleotide binding, redox states of the cofactors, and solvent condition (53)(54)(55)(56). Electron transfer between one electron-reduced CPR and cyt c is most likely prohibited due to the "closed" conformation of CPR, whereas cyt c reduction by two electron-reduced CPR is likely to be gated by domain movement of the protein.…”