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Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b 5
Author(s) -
Mortuza Gulnahar B,
Whitford David
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00870-3
Subject(s) - heme , chemistry , cytochrome , hemeprotein , stereochemistry , mutagenesis , residue (chemistry) , porphyrin , cytochrome c , site directed mutagenesis , biochemistry , mutation , enzyme , mutant , mitochondrion , gene
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 ∼9 in the bovine protein to ∼1.6 for rat cytochrome b 5 . Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu 27 and Phe 78 are located close to different parts of the porphyrin macrocycle. 1 H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b 5 extending from Gly 1 to Lys 89 suggest, in combination with NMR data acquired for other forms of cytochrome b 5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly 1 ‐Lys 89 domain of tobacco cytochrome b 5 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 b 5 where the mutation Phe 78 >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 Leu 27 >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.