Jm. Nocek et al., CYCLIC ELECTRON-TRANSFER WITHIN THE [ZN-MYOGLOBIN, CYTOCHROME B(5)] COMPLEX, Journal of the American Chemical Society, 119(9), 1997, pp. 2146-2155
To characterize the electrostatic complex formed between myoglobin (Mb
) and cytochrome bs (Febs), we have performed flash photolysis triplet
-quenching and electron-transfer (ET) measurements of the interaction
between Zn deuteroporphyrin (ZnD)-substituted Mb (sperm whale) (ZnDMb)
and Feb(5)(trypsin-solubilized, bovine) at pH values between 6 and 7.
5. For pH values between pH 6 and pH 7.5, the quenching rate constant
(Delta k) varies linearly with [Fe(3+)b(5)]. The slope (M) obtained fr
om plots of Delta k versus [Fe(3+)b(5)] is strongly dependent on pH (M
= 140 x 10(6) M(-1) s(-1) at pH 6 and M = 2.4 x 10(6) M(-1) s(-1) at
pH 7.5). The triplet decay profiles remain exponential throughout thes
e titrations. Together, these results indicate that the association co
nstant obeys the inequality, K-a less than or equal to 3000 M(-1) and
that the lower limit for the rate constant for dissociation of the (3)
DA complex of (k(off))(min) = 10(6) s(-1) at pH 6 and (k(off))(min) =
10(4) s(-1) at pH 7.5. Transient absorption measurements have shown th
at this quenching of (3)ZnDMb by Fe(3+)b(5) can be attributed to intra
complex (ZnD)-Zn-3 --> Fe3+P ET and that the transient absorbance chan
ges observed at the D-3/D isosbestic points represent the time evoluti
on of the (D(+)A(-)), [ZnD(+)Mb, Fe(2+)b(5)] intermediate, I. The long
-time behavior of the progress curves (t greater than or equal to 20 m
s) collected during a titration of Fe(3+)b(5) by ZnDMb (reverse titrat
ion protocol) is neither purely second-order nor purely first-order bu
t rather resembles a mixed-order process involving both the (D(+)A(-))
complex and its dissociated components. Modeling this data indicates
that the D(+)A(-) complex product must dissociate with a rate constant
slower than that of the precursor, DA, complex. Theoretical studies o
f the protein pair by Brownian dynamics simulations show that Mb has a
broad reactive surface which encompasses the ''hemisphere'' that incl
udes the exposed heme edge. The most stable complexes occur when bs is
bound at one of two subdomains within this hemisphere. The kinetics m
easurements and calculations taken together allow us to discuss the re
lative importance of global and local electrostatics in regulating pro
tein-protein recognition and reactivity.