Solution structure of oxidized microsomal rabbit cytochrome b₅

Abstract

Cytochrome b 5 is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the a and g meso protons. For rabbit cytochrome b 5 , the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b 5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3 J couplings, a family of 40 energyminimized conformers were obtained with average backbone rmsd (for residues 4±84) of 0.060^0.016 nm and average target function of 0.0078 nm 2 , no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5 : 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other.Keywords: cytochrome b 5 solution structure.Cytochrome b 5 (cytb5 hereafter) is a low-spin hemoprotein which acts as an electron-transfer mediator in several redox pathways, and interacts in vivo with a number of different redox partners [1]. In the process of fatty acid desaturation, cytb5 interacts with NADH cytochrome b 5 reductase [2,3]; a role of cytb5 as electron donor to cytochrome P-450 has also been proposed [4,5]. The protein is normally membrane-bound, although a soluble form is found in erythrocytes, where its physiological role is that of reducing methemoglobin [6]. The membrane-bound form may be solubilized by incubation with proteolytic enzymes, such as trypsin. The soluble fragment still binds the heme and retains protein recognition and electrontransfer capabilities [7]. The soluble fragment of cytb5 interacts in vitro with cytochrome c, the reaction between the two being fast [7]. Also thanks to the fact that the crystallographic structures of the two individual components have been available since 1971 [8,9], this complex has been an important model for the study of long-range biological electron transfer and, more generally, of the mechanisms of protein±protein recognition and interaction. The interaction between cytb5 and cytochrome c has been studied by a number of biochemical and biophysical methods (reviewed in [10]). In particular, the small size of the two proteins had allowed the investigation of their complex by NMR spectroscopy as early as 1983 [11].Cytb5 is heterogeneous in solution, as the heme, being bound to the polypeptide chain only through the bonds between the iro...

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

“…several of those in regions with regular secondary structure, which cannot be observed in CLEANEX spectra. In the case of cytochrome c, these experiments show that the amide moieties of residues 4, [17,20]. The backbone, the heme and the two axial ligands are shown.…”

“…Cytochrome b 5 is present in solution in two isomers that differ by a 180°rotation of the heme plane [30]. The ratio of the two isomers in the rabbit protein is about 9:1 [20]. Only the resonances belonging to the major isomer were analysed.…”

“…The structure of the complex is then calculated through a soft-docking approach. Here, calculations were performed using the energy-minimized average solution structures for both free cytochrome c [17] and cytochrome b 5 [20]. The calculation procedure is divided in two steps called ''searching'' and ''scoring''.…”

“…In particular, residues 24-27 belong to the part of the b-sheet of cytochrome b 5 which is at the back of the heme cavity. The other residues are located in the four helices (a 2 to a 5 [20]) making up the heme-binding pocket, with residues in helix a 4 (spanning residues 52-62) displaying the largest chemical shift changes. The N-terminal part of helices a 3 and a 5 (which comprise residues 42-49 and 64-74, respectively) are also somewhat affected by the interaction.…”

“…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.…”