A Brownian dynamics study of the interaction of Phormidium laminosum plastocyanin with Phormidium laminosum cytochrome f

Abstract

The interaction of Phormidium laminosum plastocyanin (PC) with P. laminosum cytochrome f (cyt f) was studied using Brownian dynamics (BD) simulations. Few complexes and a low rate of electron transfer were observed for wild-type PC. Increasing the positive electrostatic field on PC by the addition of a Zn(2+) ion in the neighborhood of D44 and D45 on PC (as found in crystal structure of plastocyanin) increased the number of complexes formed and the calculated rates of electron transfer as did PC mutations D44A, D45A, E54A, and E57A. Mutations of charged residues on Phormidium PC and Phormidium cyt f were used to map binding sites on both proteins. In both the presence and absence of the Zn(2+) ion, the following residues on PC interact with cyt f: D44, D45, K6, D79, R93, and K100 that lie in a patch just below H92 and Y88 and D10, E17, and E70 located on the upper portion of the PC molecule. In the absence of the Zn(2+) ion, K6 and K35 on the top of the PC molecule also interact with cyt f. Cyt f residues involved in binding PC, in the absence of the Zn(2+) ion, include E165, D187, and D188 that are located on the small domain of cyt f. The orientation of PC in the complexes was quite random in accordance with NMR results. In the presence of the Zn(2+) ion, K53 and E54 in the lower patch of the PC molecule also interact with cyt f and PC interacts with E86, E95, and E123 on the large domain of cyt f. Also, the orientation of PC in the complexes was much more uniform than in the absence of the Zn(2+) ion. The difference may be due to both the larger electrostatic field and the greater asymmetry of the charge distribution on PC observed in the presence of the Zn(2+) ion. Hydrophobic interactions were also observed suggesting a model of cyt f-PC interactions in which electrostatic forces bring the two molecules together but hydrophobic interactions participate in stabilizing the final electron-transfer-active dock.

FIGURE 1

Charged residues and electrostatic fields on Phormidium cyt f and PC. Heme (black); Y1 on cyt f and Y83 on PC (yellow); H87 on PC (green); Arg (dark blue); Lys (light blue); Glu (red); and Asp (magenta). Electrostatic field contours: +1 kT/e⁻ (blue); and −1 kT/e (red). Electrostatic fields were calculated using GRASP (Nicholls and Honig, 1991). PC residues are numbered as in Bond et al. (1999). (A) WT cyt f; (B) WT PC plus Zn²⁺ (C) WT PC minus Zn²⁺; and (D) quadruple PC mutant (D44A + D45A + E54A + D57A) minus Zn²⁺.

FIGURE 2

The number of complexes formed in BD simulations of Phormidium cyt f and Phormidium PC under different conditions. (A) The effect of mutation of anionic residues on Phormidium PC on its interaction with Phormidium cyt f. (○) WT PC minus Zn²⁺; (×) D45A-PC minus Zn²⁺; (+) D44A + D45A-PC minus Zn²; (□) WT-PC plus Zn²⁺; (•) D44A + D45A + E54A-PC minus Zn^2+.; (*) D44A + D45A + D57A-PC minus Zn²⁺; and (▪) D44A + D45A + E54A + D57A-PC minus Zn²⁺. (B) Comparison of cyt f-PC interactions of cyanobacteria with those of green algae. Conditions were as described in the Methods section. (•) Chlamydomonas cyt f interacting with Chlamydomonas PC; (▪) Phormidium cyt f interacting with Phormidium PC in the presence of Zn²⁺; (*) Phormidium cyt f interacting with Phormidium PC in the presence of Zn^{2 +} in the absence of an electrostatic field; (□) Phormidium cyt f interacting with Chlamydomonas PC; and (○) Chlamydomonas cyt f interacting with Phormidium PC + Zn²⁺. (C) The effect of mutation of cationic residues on Phormidium PC in the presence of Zn²⁺ on the number of complexes formed with Phormidium cyt f. (▪) WT-PC; (○) K6A-PC; (•) K35A- PC; (*) K46A-PC; (□) R93A-PC; and (X) R93E-PC. (D) The effect of mutations of Phormidium cyt f on its ability to interact with Phormidium PC in the presence of Zn²⁺. (▪) WT-cyt f; (□) D42A-cyt f; (•) D63A-cyt f; (*) D187A-cyt f; (○) D122A-cyt f; and (X) E95A-cyt f.

FIGURE 3

Location of the mutations on Phormidium PC. Only those mutants having the greatest effects are shown. In the absence of the Zn²⁺ ion (Table 2), results are shown for class I (greatest stimulation) and class V (greatest inhibition; in the absence of Zn²⁺ (Table 3), results are shown for class II (greatest stimulation) and classes IV and V (greatest inhibition). (Black) Y88, H92, and the Zn²⁺ (when present); (red) anionic residues; and (blue) cationic residues. (Left) −Zn²⁺ and (right) +Zn²⁺. (Top) Front and (bottom) back of the PC molecules.

FIGURE 4

Phormidium cyt f mutations. Data were taken from Tables 4 and 5. Class I (>80% WT; dark blue); class II (59–77% WT; cyan); class III (50–59% WT; green); class IV (44–48% WT; yellow); class V (<40% WT; red); and heme (black). (A) Plus Zn²⁺ and (B) minus Zn²⁺.

FIGURE 5

Heterogeneity of Phormidium cyt f-Phormidium PC BD complexes formed under different conditions. Five of the 10 complexes that were selected for detailed analysis as described in the Methods section are depicted unless otherwise indicated. The backbone ribbons were superimposed using GRASP. The heme groups on cyt f and the Cu and Zn ions as well as R88 are shown for PC. The ionic strength was 10 mM. (A) WT-PC minus Zn²⁺; (B) Phormidium cyt f-Phormidium PC complexes from Crowley et al. (2001). Twenty-five complexes were supplied by M. Ubbink. Five of these were chosen at random for display. Conditions were 10 mM sodium phosphate buffer (pH 6.0) + 1 mM Na-absorbate; (C) WT-PC plus Zn²⁺; and (D) (D42A + D43A + E52A + D57A-PC)-minus Zn²⁺.

FIGURE 6

The complex formed between Phormidium cyt f and Phormidium PC in the presence of Zn²⁺. A representative complex was selected from the 10 chosen for detailed analysis as described in the Methods section. The peptide backbone is shown as well as selected residues. The cyt f heme (black); cyt f-Y1 and PC-Y83 (yellow); Arg (dark blue); Lys (light blue); Glu (red); and Asp (magenta). Cyt f residues are labeled in red; PC residues are labeled in black.

FIGURE 7

Location of the binding sites on Phormidium cyt f and PC. The close contacts listed in Table 6 were mapped onto cyt f (A) and PC (B). Contacts found in >8 of the 10 selected complexes are shown. The cyt f heme is also shown.