Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform.
The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.
Sun
Na
N
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582, USA.
Wang
An
A
Cowley
Aaron B
AB
Altuve
Adriana
A
Rivera
Mario
M
Benson
David R
DR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D015786:Cytochromes b5 / Q000737:chemistry*; D003905:Deuteroporphyrins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000737:chemistry*; D051336:Mitochondrial Membranes / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry; D012542:Scattering, Radiation
D003905:Deuteroporphyrins; D020033:Protein Isoforms; D006418:Heme; C028839:deuteroporphyrin-IX; D015786:Cytochromes b5