Solution structure of the cytochrome P450 reductase-cytochrome c complex determined by neutron scattering

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.

Keywords: SANS; cytochrome P450; cytochrome P450 reductase; cytochrome c; electron transfer; neutron scattering; protein structural dynamics; protein structure; protein-protein interaction; solution structure; transient complex.

Figure 1.

Burst kinetics of reduction of cytochrome c on rapid mixing with NADPH-reduced CPR. Wildtype CPR (orange) or K75E/R78E/R108Q mutant CPR (blue) was mixed with cytochrome c in a stopped-flow spectrophotometer under anaerobic conditions. The baseline on mixing cytochrome c with buffer is shown in red. AU, absorbance units.

Figure 2.

Scattering envelopes of wildtype CPR and K75E/R78E/R108Q mutant CPR with atomic models superimposed together with the P(r) plots derived from the scattering data (right). In a (wildtype CPR), the model is the crystal structure of wildtype CPR (12). In b (mutant CPR), the model is one obtained from the wildtype crystal structure after a conformational search allowing motion of the residues in the flexible hinge as described under “Experimental procedures”. In both cases, the envelope and structure on the right are related to those on the left by a 90° rotation in the direction shown by the arrows. In the structural models superimposed on the scattering envelopes, the FAD and linker domains are blue, and the FMN domain is green.

Figure 3.

Fits of the scattering curve of the oxidized state of the K75E/R78E/R108Q mutant of CPR to a two-state model. The experimental data are shown as points, with error bars representing S.D., and the model fits are shown as lines. The wildtype crystal structure is used to represent the compact state, and either the model of Huang et al. (27) (a) or the structure of the ΔTGEE mutant (24) (b) is used to represent the extended state.

Figure 4.

Scattering curves of the complex between deuterated K75E/R78E/R108Q mutant CPR and cytochrome c. a, complex fully visible (70% D₂O). b, cytochrome c match-out (43% D₂O). c, K75E/R78E/R108Q mutant CPR match-out (100% D₂O). Guinier plots are shown in the insets in each case. Conditions were 100 mm BES, pH 7.0, at 15 °C with the appropriate percentage of D₂O, as indicated in parentheses, to achieve the desired contrast.

Figure 5.

Ab initio scattering envelopes (left) and intraparticle distance distributions (right) of the CPR mutant–cyt c complex. a, full complex. b, CPR mutant (cyt c match-out). c, cyt c (CPR match-out). In each case, the scattering envelope on the right is related to that on the left by a 90° rotation in the direction indicated by the arrows. In the structural models superimposed on the scattering envelopes, the FAD and linker domains of the CPR mutant are blue, the FMN domain is green, and cytochrome c is red.

Figure 6.

The model of the CPR mutant–cyt c complex that best accounts for the data. The FMN domain of the CPR mutant is shown in green, and its FAD and linker domains are in blue. cyt c is shown in red. The FMN of the CPR mutant and the heme of cyt c are shown in magenta.