Abstract
Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c. This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein–protein complexes.
Highlights
Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved
The FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of cytochrome P450 reductase (CPR)
Rapid mixing of prereduced CPR and cyt c leads to a burst of cyt c reduction, essentially within the experimental dead time of the stopped-flow spectrophotometer, by those CPR molecules that are in a reactive (“open”) state followed by a slower reduction of cyt c by those CPR molecules that exist in a cyt c– unreactive (“closed”) conformation [32]
Summary
To facilitate the study of the CPR– cyt c complex, we used the K75E/R78E/R108Q mutant of CPR in which two salt bridges stabilizing the compact conformation (see below) are abolished. These data clearly indicate a slightly more extended average structure for the mutant than for the wildtype protein in the oxidized state with a visible “cleft” in the mutant structure. That the abolition in the mutant CPR of two salt bridges stabilizing the compact conformation [27] does lead to an increase in the proportion of the extended, cyt c–reactive conformation in both the oxidized and reduced states and that this in turn leads to an increase in the amplitude of the burst phase of the reduction of cytochrome c
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