Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron.
A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.
Hay
Sam
S
Photobioenergetics, Research School of Biological Sciences, The Australian National University, Canberra ACT 0200, Australia. hay@rsbs.anu.edu.au
Wydrzynski
Tom
T
D001483:Base Sequence; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013816:Thermodynamics
D003573:Cytochrome b Group; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006418:Heme; C017850:cytochrome b562, E coli; D007501:Iron