CYCLIC ELECTRON-TRANSFER WITHIN THE [ZN-MYOGLOBIN, CYTOCHROME B(5)] COMPLEX

Citation
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
Citations number
41
Categorie Soggetti
Chemistry
ISSN journal
00027863
Volume
119
Issue
9
Year of publication
1997
Pages
2146 - 2155
Database
ISI
SICI code
0002-7863(1997)119:9<2146:CEWT[C>2.0.ZU;2-P
Abstract
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.