title: Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b5.
abstract: A comparison of the primary sequences of the heme binding domains of bovine and rat microsomal cytochrome reveal differences at only six residues. These residues must therefore provide the origin for the observed variation in the ratio of the heme orientational isomers, the equilibrium constant of which ranges from approximately 9 in the bovine protein to 1.6 for rat cytochrome b5. Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu27 and Phe78 are located close to different parts of the porphyrin macrocycle. 1H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b5 extending from Gly1 to Lys89 suggest, in combination with NMR data acquired for other forms of cytochrome b5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly1-Lys89 domain of tobacco cytochrome b5 has two equally abundant heme orientational isomers but retains the leucine side chain at position 27 whilst phenylalanine 78 is replaced by tyrosine. A more direct role for residue 78 in modulating the heme ratio is shown by site directed mutagenesis of bovine microsomal cytochrome b5 where the mutation Phe78 > Tyr shifts the equilibrium constant for the heme orientational isomers from 9 to 3.5. Whilst the ratio is clearly shifted towards that exhibited by the rat protein the incomplete transition suggested the involvement of other residues. The mutation of Leu27 > Val was shown to result in a slightly smaller change in ratios of each isomer (from 9 to 4.0). Together these results point to the importance of these residues in modulating the ratio of heme isomers.
authors.affiliation: Laboratory of Structural Biochemistry, Queen Mary and Westfield College, London, UK.
authors.forename: G B
authors.initials: GB
authors.lastname: Mortuza
authors.forename: D
authors.initials: D
authors.lastname: Whitford
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics*; D006418:Heme / Q000737:chemistry* / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010947:Plants, Toxic; D051381:Rats; D014026:Nicotiana; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D006418:Heme; D010649:Phenylalanine; D015786:Cytochromes b5