The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precurso r protein lacking the C-terminal 33 amino-acid residues. The protein is ide ntical [as judged by H-1-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f puri fied from turnip leaves. Several residues in the hydrophobic patch surround ing the haem group have been changed by site-directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor c hanges in the plastocyanin-binding constant K-a and the second-order rate c onstant for electron transfer to plastocyanin, whereas the Y160S mutant sho wed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an incre ased driving force due to a 27-mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be as cribed to an increased binding constant and a 14-mV decrease in midpoint re dox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the bi nding constants and altering the midpoint redox potential of the cytochrome haem group.