A Brownian Dynamics computational study of the interaction of spinach plastocyanin with turnip cytochrome f: the importance of plastocyanin conformational changes.

Brownian Dynamics (BD) computer simulations were used to study electrostatic interactions between turnip cytochrome f (cyt f) and spinach plastocyanin (PC). Three different spinach PC structures were studied: The X-ray crystal structure of Xue and coworkers [(1998) Protein Sci 7:2099-2105] and the NMR structure of Musiani et al. [(2005) J Biol Chem 280:18833-18841] and Ubbink and co-workers [(1998) Structure 6:323-335]. Significant differences exist in the backbone conformation between the PC taken from Ubbink and coworkers and the other two PC structures particularly the regions surrounding G10, E59-E60, and D51. Complexes formed in BD simulations using the PC of Ubbink and colleagues had a smaller Cu-Fe distance than the other two. These results suggest that different PC conformations may exist in solution with different capabilities of forming electron-transfer-active docks. All three types of complexes show electrostatic contacts between D42, E43, and D44 on PC and K187 on cyt f as well as between E59 on PC and K58 on cyt f. However, the PC of Ubbink and coworkers reveals additional contacts between D51 and cyt f as a result of the difference in backbone configuration. A second minor complex component was observed for the PC of Ubbink and co-workers and Xue and co-workers which had contacts between K187 on cyt f and E59 and E60 on PC rather than between K187 on cyt f and D42-D44 on PC as observed for the major components. This second type of complex may represent an earlier complex which rearranges to form a final complex capable of electron transfer.

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.

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.

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins.

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.

A Brownian dynamics study of the effects of cytochrome f structure and deletion of its small domain in interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii.

The availability of seven different structures of cytochrome f (cyt f) from Chlamydomonas reinhardtii allowed us, using Brownian dynamics simulations, to model interactions between these molecules and their redox partners, plastocyanin (PC) and cytochrome c6 (cyt c6) in the same species to study the effect of cyt f structure on its function. Our results showed that different cyt f structures, which are very similar, produced different reaction rates in interactions with PC and cyt c6. We were able to attribute this to structural differences among these molecules, particularly to a small flexible loop between A-184 and G-191 (which has some of the highest crystallographic temperature factors in all of the cyt f structures) on the cyt f small domain. We also showed that deletion of the cyt f small domain affected cyt c6 more than PC, due to their different binding positions on cyt f. One function of the small domain in cyt f may be to guide PC or cyt c6 to a uniform dock with cyt f, especially due to electrostatic interactions with K-188 and K-189 on this domain. Our results could serve as a good guide for future experimental work on these proteins to understand better the electron transfer process between them. Also, these results demonstrated the sensitivity and the power of the Brownian dynamics simulations in the study of molecular interactions.

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants.

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.

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

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.

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

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.