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

Rui Huang,Meng Zhang,Freeborn Rwere,Lucy Waskell,Ayyalusamy Ramamoorthy

doi:10.1074/jbc.m114.582700

Rui Huang, Meng Zhang + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m114.582700

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Abstract

Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.

Highlights

Cytochrome P450 reductase (CPR) is an electron donor for many proteins
Lys-13 of cyt c locates in close proximity of the heme and forms a salt bridge with Asp-208 of FMN binding domain (FBD) in the complex model
Combined kinetic and structural information presented in this study provides insights into the function of CPR and will facilitate further understanding of the mechanistic details on how electron transfer occurs from CPR to its redox partners

Summary

Background

Cytochrome P450 reductase (CPR) is an electron donor for many proteins. Results: The FMN binding domain (FBD) of CPR interacts with cytochrome c transiently and reduces cytochrome c at a higher rate than CPR. Antibody binding studies revealed that the antibody designed to bind to the segment from Gly-204 to Gly-218 of CPR was not able to abrogate the CPR-cyt c interaction [19] Mutation of another acidic cluster, including Asp-113, Glu-115, and Glu-116 on the ␤2-␣4 coil of CPR, improves its cyt c reducing activity, facilitating the formation of a more functional complex [13]. In our study we utilize solution NMR techniques to probe the interaction between CPR and cyt c, which can allow us to identify residues on the binding interface and further predict electron transfer pathway between the two proteins. To simplify the electron transfer process and focus our study on the interprotein interaction, we decided to investigate the electron transfer and complex formation between the isolated FMN binding domain, which is the direct electron donor to cyt c. The structural models generated by high ambiguity driven biomolecular docking (HADDOCK) based on NMR restraints suggest potential electron transfer pathways that provide strong electronic coupling between the redox centers

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION