A Brownian dynamics study of the interactions of the luminal domains of the cytochrome b6f complex with plastocyanin and cytochrome c6: the effects of the Rieske FeS protein on the interactions

Abstract

The availability of the structures of the cytochrome b6f complex (cyt b6f), plastocyanin (PC), and cytochrome c6 (cyt c6) from Chlamydomonas reinhardtii allowed us, for the first time, to model electron transfer interactions between the luminal domains of this complex (including cyt f and the Rieske FeS protein) and its redox partners in the same species. We also generated a model structure in which the FeS center of the Rieske protein was positioned closer to the heme of cyt f than observed in the crystal structure and studied its interactions with both PC and cyt c6. Our data showed that the Rieske protein in both the original crystal structure and in our modeled structure of the cyt b6f complex did not physically interfere with binding position or orientation of PC or cyt c6 on cyt f. PC docked on cyt f with the same orientation in the presence or the absence of the Rieske protein, which matched well with the previously reported NMR structures of complexes between cyt f and PC. When the FeS center of the Rieske protein was moved close to the heme of cyt f, it even enhanced the interaction rates. Studies using a cyt f modified in the 184-191 loop showed that the cyt f structure is a more important factor in determining the rate of complex formations than is the presence or the absence of the Rieske protein or its position with respect to cyt f.

FIGURE 1

Distances between the Rieske FeS cluster and the cyt f heme in the crystal structure (cyt f+Rieske subcomplex-far) and the close model structure (cyt f+Rieske subcomplex-close) in the C. reinhardtii cyt b₆f complex. All of the distances are in Angstroms.

FIGURE 2

Interactions between the cyt f+Rieske subcomplex-far with PC (A) and cyt c₆ (B) at 10 mM ionic strength and pH 7.0. Five sets of 10,000 trajectories each were carried out, after which the average of the number of the complexes formed at the closest approach of each trajectory was plotted as a function of the Fe-Cu distance for PC interactions and heme-heme distance for cyt c₆ interactions. The number of complexes with closest metal-to-metal distances between 15.25 and 15.5 Å is shown on the abscissa and plotted at 15.5 Å. See the Methods sections for a description of the cyt f-modified complexes.

FIGURE 3

Interactions between the cyt f+Rieske subcomplex-close with PC (A) and cyt c₆ (B) at 10 mM ionic strength and pH 7.0. The conditions are the same as for Fig. 2. For a description of the close model and the cyt f-modified complexes, see Methods section.

FIGURE 4

Overlay of the cyt f structure B from the PDB code 1CFM on the extramembrane domain of the cyt f subunit from the cyt b₆f complex (PDB code 1Q90). The 1CFM-B cyt f backbone and all of its basic residues were colored by their temperature factors (β-factors), in which the molecule is colored from dark blue for low β-factors to red for high β-factors (the heme is colored red for visualization purpose). The backbone and all of the basic residues of the 1Q90 cyt f are shown in gray, and its heme is in black. As can be seen, the loop of residues A-184–G-191 of the 1CFM-B cyt f has some of the highest β-factors compared to the rest of the molecule (indicating its high flexibility) and is oriented very differently than the 1Q90 loop. The key basic residues in both cyt fs are labeled. This figure was taken from Haddadian and Gross (38) and generated by the program Deep View (40).

FIGURE 5

Electrostatic fields of reduced cyt f (A), reduced cyt f plus oxidized Rieske (B) in the cyt f+Rieske subcomplex-far, and reduced cyt f plus oxidized Rieske in the cyt f+Rieske subcomplex-close (C) from the C. reinhardtii cyt b₆f complex. The electrostatic field contours at +1 (blue) and –1 (red) were calculated at 10 mM ionic strength and pH 7.0 using the program GRASP (41). The heme and the FeS cluster are shown as space-filling models. The backbone of cyt f is colored in black and that of the Rieske protein in green.

FIGURE 6

Final positions of the center of mass of PC molecules with respect to cyt f (A) and the cyt f+Rieske subcomplex-far (B) for 400 different trajectories. The complexes formed at the closest approach of each trajectory with the Fe-Cu distances ≤90 Å were shown. The program MacroDox outputs the structure of the complexes formed in the form of a PDB file and replaces the PC molecules with their centers of mass. The cyt f and Rieske molecules are shown as space-filling models and are colored as gray and green, respectively. The centers of mass of PC molecules are shown as red spheres. The heme is in black. The five important lysine residues (Lys-58, 65, 66, 188, and 189) on cyt f are shown as blue.

FIGURE 7

Overlays of the peptide backbones of five complexes each, for the cyt f+Rieske subcomplex-far (A) and the cyt f+Rieske subcomplex-close (B) with PC. These complexes were chosen at random from those complexes with the Fe-Cu distances less than the peak distances in the plots of the complexes formed. The overlays were constructed using the program GRASP (41). The heme and the Cu atoms are shown as space-filling models and are in black.