The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.

Abstract

Site‐directed mutants of the pea plastocyanin gene in which the codon for the surface‐exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild‐type plastocyanin by 1H‐NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped‐flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8‐fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40‐fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.

Dynamic Interaction of Plastocyanin with the Cytochrome bf Complex

“…At 25°C the value of the rate constant is 62 ϫ 10 6 M Ϫ1 s Ϫ1 in the presence of 150 mM NaCl. Assuming the same ionic strength dependence of the reaction rate at 5 (see below) and 25°C, this value extrapolates to a rate constant of 185 ϫ 10 6 M Ϫ1 s Ϫ1 at 90 mM NaCl, which is by a factor of 4 faster than that reported for truncated cyt f under these conditions (24,25,27) but comparable to the value of 100 ϫ 10 6 M Ϫ1 s Ϫ1 found recently (29). To analyze the surface regions of plastocyanin involved in the rate-limiting step of the reaction with the cyt bf complex, the role of residues in the hydrophobic and acidic patches of spinach plastocyanin was probed using site-directed mutagenesis.…”

“…A substitution of Leu-12 in the hydrophobic patch (25) by Asn and Glu was found to accelerate and slow down the second-order rate constant, respectively, a finding that is not fully understood but may suggest a role of this site. For the interaction with truncated, soluble cyt f, it is widely accepted that the electron is transferred to the copper center of plastocyanin via Tyr-83 (20,24,25,27), which seems to be favored by the high electron density at the thiolate of the copper ligand Cys-84 (31).…”

Engineering type 1 copper sites in proteins

“…4) acidic patch. This patch functions as the docking site for cytochrome f and there are indications that electrons may reach the Cu via Tyrs3 and CYSTS, the latter being a ligand of the Cu [77,78]. It has been estimated that this path is 10 times less efficient than the electron transfer path through His*' which is used by plastocyanin when it has to give off its electron to PTm+, the oxidized reaction centre of photosystem I [79].…”

Analysis of the 1H‐NMR Chemical Shifts of Cu(I)‐, Cu(II)‐ and Cd‐Substituted Pea Plastocyanin

“…Also the reaction of plastocyanin with cytochrome c, acting as a non-physiological substitute for cytochromef, has been studied extensively. From this work it has emerged that PSI probably binds to both the northern and eastern sides of the molecule but reacts via the hydrophobic patch (Anderson et al, 1987;Takabe and Ishikawa, 1989;Nordling et al, 1991;Hervas et al, 1995), while cytochrome f and c bind and react via the acidic patch (Niwa et al, 1980;Augustin et al, 1983;Beoku-Betts et al, 1985;King et al, 1985;Anderson et al, 1987;Morand et al, 1989;Takabe and Ishikawa, 1989;Gross and Curtiss, 1991 ;He et al, 1991 ;Roberts et al, 1991 ;Christensen et al, 1992;Modi et al, 1992a,b;Meyer et al, 1993). The latter reaction complexes appear not to be static; KostiC and co-workers have tound evidence that a fast rearrangement of the partners within the cytochrome .…”