Transient absorbance measurements following laser
flash photolysis have been used to measure the rate constants
for electron transfer (et) from reduced Anabaena
ferredoxin (Fd) to wild-type and seven site-specific charge-reversal
mutants of Anabaena ferredoxin:NADP+
reductase (FNR). These mutations have been designed to
probe the importance of specific positively charged amino
acid residues on the surface of the FNR molecule near the
exposed edge of the FAD cofactor in the protein–protein
interaction during et with Fd. The mutant proteins fall
into two groups: overall, the K75E, R16E, and K72E mutants
are most severely impaired in et, and the K138E, R264E,
K290E, and K294E mutants are impaired to a lesser extent,
although the degree of impairment varies with ionic strength.
Binding constants for complex formation between the oxidized
proteins and for the transient et complexes show that the
severity of the alterations in et kinetics for the mutants
correlate with decreased stabilities of the protein–protein
complexes. Those mutated residues, which show the largest
effects, are located in a region of the protein in which
positive charge predominates, and charge reversals have
large effects on the calculated local surface electrostatic
potential. In contrast, K138, R264, K290, and K294 are
located within or close to regions of intense negative
potential, and therefore the introduction of additional
negative charges have considerably smaller effects on the
calculated surface potential. We attribute the relative
changes in et kinetics and complex binding constants for
these mutants to these characteristics of the surface charge
distribution in FNR and conclude that the positively charged
region of the FNR surface located in the vicinity of K75,
R16, and K72 is especially important in the binding and
orientation of Fd during electron transfer.