Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

Abstract

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.

FIGURE 1

Electric field representation of Chlamydomonas cyt f (Structure B of 1CFM). The electrostatic field contour at +1 kT/e⁻ (gray) was calculated at 10 mM ionic strength, pH 7.

FIGURE 2

Electrostatic fields of Chlamydomonas (A) PC and (B) cyt c₆. The electrostatic field contour at −1 kT/e⁻ is shown (gray). Other conditions were as for Fig. 1.

FIGURE 3

Brownian dynamics simulations. PC or cyt c₆ is randomly placed on a sphere (B) of radius 86 Å (PC) and 85 Å (cyt c₆) distance from the center of mass of cyt f . It is allowed to move one step under the influence of an electrostatic field (EL) and a random Brownian factor (BR). Many such steps form a trajectory that is terminated when PC or cyt c₆ exits sphere D (200 Å). The smallest metal to metal distance attained in a trajectory (point C) is recorded for each trajectory. At that point, the structure of the complex formed, the fifteen closest electrostatic contacts and the electrostatic interaction energy is also recorded.

FIGURE 4

MacroDox simulations of the interaction of cyt f with PC and cyt c₆. (A) The interaction of Chlamydomonas cyt f with PCs from Chlamydomonas, spinach, and Anabaena as well as azurin. Chlamydomonas cyt f-Chlamydomonas PC (▪); turnip cyt f-spinach PC (•); Chlamydomonas cyt f-spinach PC (Δ); turnip cyt f-spinach PC (∇); Chlamydomonas cyt f-Chlamydomonas PC-no electrostatic field (□); Chlamydomonas cyt f-Anabaena PC (▵); Chlamydomonas cyt f-azurin (○). (B) The interaction of Chlamydomonas cyt f with Chlamydomonas PC and cyt c₆. Five sets of 1000 trajectories each were carried out at 10 mM ionic strength (pH 7.0) after which the number of successful complexes was plotted as a function of Cu-Fe (PC, azurin) or Fe-Fe (cyt c₆) distance at closest approach. The number of complexes with distances of closest metal to metal distance rounded to the next highest Å. Other conditions were as described in the Methods section.

FIGURE 5

The structure of Chlamydomonas cyt f-PC and Chlamydomonas cyt f-cyt c₆ complexes. (A) A typical cyt f-PC complex from MacroDox simulations. (B) The turnip cyt f-spinach PC complex model 1 of Ubbink et al. (1998) (blue) superimposed on that of the Chlamydomonas cyt f-PC BD complex depicted in A (red). The two molecules were superimposed using Deep View from Swiss Prot (Guex and Peitsch, 1997; http://www.expasy.ch/spdbv/) aligning of residues 1–171 of the large domains of the cyt f molecules. (C) A typical cyt f-cyt c₆ complex from MacroDox simulations. Representative BD complexes were taken from those used to construct Table 1. See the Methods section for the manner in which the complexes were chosen and displayed. Color codes for A and C were as follows: Heme, black; histidine, green; tyrosine, yellow; arginine, dark blue; lysine, light blue; glutamate, red; asparatate, magenta.

FIGURE 6

Locations of the binding site(s) on cyt f for PC and cyt c₆. Binding site for cyt c₆ alone: gray with label in italics; binding site for both PC and cyt c₆: black.

FIGURE 7

Orientation of complexes. (A) Cyt f-PC complexes. (B) Cyt f-cyt c₆ complexes. (C) K189A-cyt f-PC complexes. (D) K189E-cyt f-PC complexes. (E) Wild-type cyt f at 300 mM ionic strength. Five complexes were chosen for each condition and were superimposed using GRASP. The protein backbones, the cyt f heme and the PC Cu atoms are shown. The ionic strength is 10 mM except where indicated.

FIGURE 8

The effect of cyt f mutants on the formation of cyt f-PC complexes at 10 mM ionic strength. (A) The number of complexes formed as a function of Cu-Fe distance for wild-type and cyt f mutants. Other conditions were as for Fig. 4. (B) Position of the mutants on cyt f. Inhibition of complex formation: Class I, ≤30% (R88, K94, K110, K164, and K165), light gray; Class II, 30–60% (K58, K66, K121, K122, R156, and R207) dark gray with labels in italics; Class III, > 60% (K65, K188, and K189), black with labels underlined.