Insights into the role of the histidines in the structure and stability of cytochrome c.
In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.
Sinibaldi
Federica
F
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata, via Montpellier 1, 00133 Rome, Italy.
Howes
Barry D
BD
Piro
M Cristina
MC
Caroppi
Paola
P
Mei
Giampiero
G
Ascoli
Franca
F
Smulevich
Giulietta
G
Santucci
Roberto
R
D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D013059:Spectrum Analysis, Raman
D005656:Fungal Proteins; D006639:Histidine; D045304:Cytochromes c; D008239:Lysine