Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c.
A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.
Lu
Y
Y
Beckman Institute, California Institute of Technology, Pasadena 91125.
Casimiro
D R
DR
Bren
K L
KL
Richards
J H
JH
Gray
H B
HB
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003580:Cytochromes / Q000096:biosynthesis / Q000378:metabolism*; D045304:Cytochromes c; D004926:Escherichia coli; D006418:Heme / Q000032:analysis; D006736:Horses; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D003580:Cytochromes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D045304:Cytochromes c; D008715:Methionine; D000409:Alanine