NMR Studies of the Association of Cytochrome b₅ with Cytochrome c

Abstract

In an effort to gain greater insight into the molecular mechanism of the electron-transfer reactions of cytochrome b(5), the bovine cytochrome b(5)-horse cytochrome c complex has been investigated by high-resolution multidimensional NMR spectroscopy using (13)C, (15)N-labeled cytochrome b(5) expressed from a synthetic gene. Chemical shifts of the backbone (15)N, (1)H, and (13)C resonances for 81 of the 82 residues of [U-90% (13)C,U-90% (15)N]-ferrous cytochrome b(5) in a 1:1 complex with ferrous cytochrome c were compared with those of ferrous cytochrome b(5) in the absence of cytochrome c. A total of 51% of these residues showed small, but significant, changes in chemical shifts (the largest shifts were 0.1 ppm for the amide (1)H, 1.15 for (13)C(alpha), 1.03 ppm for the amide (15)N, and 0.15 ppm for the (1)H(alpha) resonances). Some of the residues exhibiting chemical shift changes are located in a region that has been implicated as the binding surface to cyt c [Salemme, F. R. (1976) J. Mol. Biol. 10, 563-568]. Surprisingly, many of the residues with changes are not located on this surface. Instead, they are located within and around a cleft observed to form in a molecular dynamics study of cytochrome b(5) [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693](.) The rim of this cleft can readily accommodate cytochrome c. Molecular dynamics simulations of the Salemme and cleft complexes were performed for 2 ns and both complexes were stable.

A further investigation of the cytochrome b 5–cytochrome c complex

“…All NMR spectrometers were equipped with 1 H, 13 C, 15 N TXI probes with z-pulsed field gradients. Cytochrome b 5 is present in solution in two isomers that differ by a 180°rotation of the heme plane [30].…”

“…Sensitivity improved, echo-anti-echo HSQC experiments [33,34] were acquired to follow 1 H and 15 N chemical shift changes throughout the titrations. 15 N R 1 and R 2 relaxation rates were measured using available pulse sequences at 11.7 T (50 MHz for 15 N Larmor frequency), through, respectively, inversion-recovery [35] and CPMG measurements [36]. In all experiments, solvent suppression was achieved with the water flip-back scheme, which avoids water saturation [37].…”

“…This hypothesis was strongly criticized [4] and a 1:1 complex was then nearly always assumed. Later, as 15 N-enriched cytochrome b 5 became available, heteronuclear single quantum correlation spectroscopy (HSQC) experiments were performed in the presence of cytochrome c [14,15] in an attempt to characterize the interaction surface of cytochrome b 5 . As a result of this large amount of data from different sources, a number of similar models for the interaction have been proposed in the literature as well as the possibility of multiple binding sites.…”

“…A 1 H-15 N NMR investigation of cytochrome c interacting with cytochrome b 5 is lacking because, until recently, it was difficult to express cytochrome c in conditions and with yields suitable to label it with 15 N. The recent availability of protocols for cytochrome c expression in Escherichia coli [16], enabling the correct covalent attachment of the heme cofactor to the protein frame, prompted us to re-examine the cytochrome b 5 -cytochrome c complex. The system was studied with both proteins in the reduced state since it was impossible for us to keep one or both proteins in the oxidized state for the long time of the NMR experiments.…”

“…The characterization of these adducts still has a biological meaning, as the surfaces and their electrostatic potentials are not affected by the redox state (as witnessed by the comparison of the structures in the two states [17,18,19,20]) and therefore are expected to provide relevant information about the adducts. 15 N chemical shifts were monitored through 1 H-15 N HSQC experiments, which are very sensitive and thus relatively quick. The characterization of the interaction between the proteins and the solvent provided information complementary to that of chemical shift mapping.…”