To characterize the electrostatic complex formed between myoglobin (Mb) and cytochrome b5 (Feb5), 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 Feb5(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 (Δk) varies linearly with [Fe3+b5]. The slope (M) obtained from plots of Δk versus [Fe3+b5] is strongly dependent on pH (M = 140 x 106 M-1 s-1 at pH 6 and M = 2.4 x 106 M-1 s-1 at pH 7.5). The triplet decay profiles remain exponential throughout these titrations. Together, these results indicate that the association constant obeys the inequality, K(a) ≤ 3000 M-1 and that the lower limit for the rate constant for dissociation of the 3DA complex of (k(off))(min) = 106 s-1 at pH 6 and (k(off))(min) = 104 s-1 at pH 7.5. Transient absorption measurements have shown that this quenching of 3ZnDMb by Fe3+b5 can be attributed to intracomplex 3ZnD → Fe3+P ET and that the transient absorbance changes observed at the 3D/D isosbestic points represent the time evolution of the (D+A-), [ZnD+Mb, Fe2+b5] intermediate, I. The long-time behavior of the progress curves (t ≤ 20 ms) collected during a titration of Fe3+b5 by ZnDMb (reverse titration protocol) is neither purely second-order nor purely first-order but 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 of the protein pair by Brownian dynamics simulations show that Mb has a broad reactive surface which encompasses the 'hemisphere' that includes the exposed heme edge. The most stable complexes occur when b5 is bound at one of two subdomains within this hemisphere. The kinetics measurements and calculations taken together allow us to discuss the relative importance of global and local electrostatics in regulating protein-protein recognition and reactivity.
ASJC Scopus subject areas
- Colloid and Surface Chemistry