Conversion of cytochrome b562 to c-type cytochromes.
Cytochrome b562 from the periplasm of Escherichia coli is the only member of a family of cytochromes sharing the 4-alpha-helical bundle structural motif that does not have a covalently bound heme. We have introduced cysteine residues into the amino acid sequence of cytochrome b562 in positions homologous to those found in the other members of the family, generating the ubiquitous heme-binding peptide (-C-X-Y-C-H-) found in virtually all c-type cytochromes. The resulting single-cysteine-containing mutants, R98C and Y101C, together with the double mutant combining both of these mutations have been expressed into the periplasm of E. coli. The apo- and holoprotein products of each mutation have been isolated, and all the mutants produce multiple species with covalently attached heme. Results from ion exchange chromatograph, optical spectroscopy, SDS gel electrophoresis, and electrospray mass spectrometry identified those species that appear to be cytochrome b562 holoprotein with thioether covalent linkages to the heme as the only difference in chemical composition between them and the wild-type protein. Results from 1H-NMR experiments prove the existence of the expected c-type covalent bonds in each of these proteins and show that the structure of the heme pocket is not significantly perturbed by the covalent modification(s). These proteins all have perturbed optical spectra, compared with those of the wild-type protein, that are consistent with the modifications but are still characteristic of six-coordinate, low-spin cytochromes with Met-His ligation to the heme iron in both oxidation states.
Barker
P D
PD
Laboratory of Molecular Biology, MRC Centre, Cambridge, U.K.
Nerou
E P
EP
Freund
S M
SM
Fearnley
I M
IM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003198:Computer Simulation; D003545:Cysteine; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D006418:Heme; C017850:cytochrome b562, E coli; D003545:Cysteine