Probing the Differences between Rat Liver Outer Mitochondrial Membrane Cytochrome b₅ and Microsomal Cytochromes b₅

Abstract

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR...

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

“…cyt b 5 is an amphiphatic protein consisting of two domains: an N-terminal water-soluble heme-binding domain and a C-terminal hydrophobic membrane-anchoring domain (32). Mammalian genomes encode two such proteins, one being anchored to the membrane of the endoplasmic reticulum and the other being anchored to the outer membrane of mitochondria (33). To improve the solubility and thus the expression and purity of the mitochondrial cyt b 5 protein used in this study, the protein was partially truncated at the C terminus to remove the hydrophobic membrane-anchoring domain.…”

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

“…There are two membrane-bound isoforms of CYB5, the microsomal (CYB5A) and the mitochondrial (CYB5B) isoform, which both derive from two different genes (19,20). CYB5A and CYB5B show high sequence and structural similarities (20,38).…”

“…Like the flavoprotein CYB5R, CYB5 can be found as an integral membrane protein in the endoplasmic reticulum and the outer mitochondrial membrane (OMM), as well as a soluble form in erythrocytes (19,20). In contrast to CYB5R, two separate genes encode the different isoforms of CYB5, with CYB5A encoding the microsomal and soluble isoform and CYB5B encoding the mitochondrial isoform.…”

Assignment of the Ferriheme Resonances of the High-Spin Forms of Nitrophorins 1 and 4 by 1H NMR Spectroscopy: Comparison to Structural Data Obtained from X-ray Crystallography

“…The poor resolution of the 1 H NMR spectra of NP1 and NP4 is caused by the existence of nearly equal amounts of the two heme orientational isomers, the well-known "heme rotational disorder" observed in most other heme b-containing proteins. 43,47,[53][54][55][56][57][58][59][60][61] This was shown by titrating the nitrophorin apoproteins with the "symmetrical hemin," trivial name 2,4-dimethyldeuterohemin IX, i.e., 1,2,3,4,5,8-hexamethyl,6,7-(dipropionic acid) porphyrinatoiron(III). Figure 2 shows the 1 H NMR spectra of these high-spin forms of the four proteins.…”