The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro

Abstract

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f±plastocyanin complex determined by NMR.

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

“…The previously reported mutational analyses, cross-linking studies, and ion strength dependencies of the Pc-PSI and Pc-cyt b 6 f electron transport rates have shown that the acidic patch residues of Pc and the basic residues of PSI and cyt b 6 f are responsible for the Pc-PSI (Hippler et al, 1989(Hippler et al, , 1997Lee et al, 1995;Sigfridsson et al, 1996Sigfridsson et al, , 1997Young et al, 1997) and Pc-cyt b 6 f electron transport reactions (Modi et al, 1992b;Lee et al, 1995;Hippler et al, 1998;Gong et al, 2000b;Illerhaus et al, 2000). Therefore, the acidic patch of Pc may increase its local concentration around PSI and cyt b 6 f, thus facilitating the rapid formation of the electron transfer complex by occasionally forming dynamic salt bridges (Bergkvist et al, 2001) or long-distance ion pairs (Figure 7, left).…”

“…Mutational and cross-linking studies of the PSI from C. reinhardtii revealed that two Trp residues close to P700 stemming from the PsaA and PsaB subunits, PsaA-Trp-651 and PsaB-Trp-627 in Chlamydomonas reinhardtii (Sommer et al, 2002(Sommer et al, , 2004, and clusters of basic residues located adjacent to the Trp residues from the PsaF subunit, PsaF-Lys-16, PsaF-Lys-23, and PsaF-Lys-30 in C. reinhardtii (Hippler et al, 1989(Hippler et al, , 1997(Hippler et al, , 1998, participate in the electron transport (Busch and Hippler, 2011). The cyt b 6 f from eukaryotic plants also contains several hydrophobic residues surrounding the heme in cyt f and clusters of basic residues located adjacent to the hydrophobic residues, and these residues also participate in electron transport, based on mutational analyses of cyt b 6 f and isolated cyt f (Soriano et al, 1996(Soriano et al, , 1998Gong et al, 2000aGong et al, , 2000b. However, the roles of these residues in the rapid turnover of Pc remain unknown.…”

FamClash: A method for ranking the activity of engineered enzymes

“…FamClash is motivated by the method developed by Govindarajan et al (29) that encompasses sequence information from not only the parental sequences but also from members composing the entire protein family to be engineered. In addition, because many studies have shown that the interactions of even distal residues can have a significant impact on the activity of the hybrids (36)(37)(38), we include such noncontacting pairs in our analysis. FamClash proceeds in three steps: (i) pairs of positions in the protein family sequence alignment are iden-tified for which a particular property triplet of charge, volume, and hydrophobicity is preferentially retained; (ii) residue pairs at these positions in the hybrids are examined to check whether they retain the properties observed in the protein family; and (iii) ranking these hybrids with respect to their probable activity based on the extent of departure from the family sequences, measured in terms of number of clashes.…”

Electron Transfer and Stability of the Cytochromeb6 f Complex in a Small Domain Deletion Mutant of Cytochrome f

“…However, observations from experiments with in vitro systems yielded different conclusions. The results from the electron transfer experiments carried out in vitro (17,20,21) and in nebulized cells (16) and from molecular dynamic modeling (37)(38)(39) and the solution structure of the complex between cytochrome f and plastocyanin (40,41) provided evidence that the lysine residues from the small domain do interact with plastocyanin. This discrepancy between in vivo and in vitro behaviors remains unresolved.…”

“…A lysine patch was found in cytochrome f (5-7) from higher plants and C. reinhardtii and was postulated to be involved in the interaction with plastocyanin (5). Mutations within the large domain generated interference with the electron transfer (13)(14)(15)(16)(17)(18)(19)(20)(21), the proton translocation (18), and the maturation of cytochrome f (13,19). When mutants were isolated in which heme attachment was altered in the large domain of cytochrome f, the assembly of the cytochrome b 6 f complex and the insertion of the heme group were disrupted (13).…”