The identification of cation‐binding domains on the surface of microsomal cytochrome b₅ using ¹H‐NMR paramagnetic difference spectroscopy

Abstract

One-dimensional and two-dimensional 'H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethy1enediamine) chromium(II1) [Cr(en)z ' 1 to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites I1 and 111 are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)z' and cytochrome h5 using two-dimensional double-quantumfiltered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en); ' broadens many cytochrome h, resonances including the haem propionates, His26, Ala54, Thr55 and His8O. Although the pattern of line-broadening of resonances at sites 11 and I11 is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.Cytochrome h5, an amphipathic protein located in the endoplasmic reticulum of cells, participates in an NADHlinked fatty acid desaturation pathway and an NADPHdriven P-450 hydroxylation system [1, 21. Domains showing considerable sequence similarity to the microsomal protein have also been recognised in erythrocytes, as part of the metHb reductase system, and in many mitochondrial enzymes such as sulphite oxidase [3 -71. Despite the comparatively common occurrence of these 'b5-like' domains in cells, there remains a gap in our understanding of how such proteins perform rapid and specific long-range electron transfer with other redox partners. One approach to this problem is to unravel how the microsomal protein associates with positively charged molecules.Knowledge concerning the interaction of microsomal cytochrome h5 with oppositely charged redox partners has been advanced by the crystallisation and subsequent structural resolution of the soluble domain of this protein [8]. This hydrophilic domain of cylindrical shape contains six CI helices and five / I strands arranged around a porphyrin ring that is Correspondence to D. Whitford, Dept of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, EnglandAbbreviations. Cr(en)z', tris(ethy1enediamine) chromium(Il1); Co(en): +, tris(cthy1enediamine) cobalt(II1); DQF-COSY, doublequantum-filtered c...

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

“…3A). The existence of different interaction regions on cytochrome b 5 has already been proposed [15,57]. The above findings are also in agreement with the diffusional properties of the molecules in the adduct, obtained from 15 N relaxation data using well-documented approaches [38,39,58].…”

“…The second interaction region appears instead to be more susceptible to variations in primary sequence. In our case, the most likely additional binding site comprises helix a 5 (55)(56)(57)(58)(59)(60)(61) and the turn up to residue 64.…”

“…3A), as both studies find the same region. This comprises helix a 4 (43)(44)(45)(46)(47)(48), part of helix a 5 (55)(56)(57)(58) and part of the strand formed by residues 23-27. The second interaction region appears instead to be more susceptible to variations in primary sequence.…”

Nuclear magnetic resonance of paramagnetic metalloproteins

“…In the DQF-COSY experiments, the only cross peaks substantially altered by complex formation were those attributable to the heme propionates of cytochrome b5. In the binary complex between ferricytochrome b5 and ferricytochrome c , Cr(en)33+ broadens many cytochrome b5 resonances. 526 Cytochrome c shows significant line broadening of resonances in the presence of the complex. Although the pattern of line broadening of resonances at sites I1 and I11 is unaltered by complex formation, cytochrome c selectively shields some residues at site I, the heme edge site.…”

“…One-and two-dimensional lH NMR methods have defined cation-binding domains on the surface of the tryptic fragment of microsomal b6. 526 The addition of Cr(en)33+ to solutions of ferricytochrome b5 reveals at least three distinct sites (indicated as I, 11, and 111) on the surface of the protein to which highly charged cations may bind. Site I contains the exposed 6-propionate group and a series of carboxylate residues.…”