title: Tuning the reduction potential of engineered cytochrome c-553.
abstract: Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 +/- 5 mV for the wt to +88 +/- 5 and +105 +/- 5 mV for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (n(B)), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 A(2) for the wt to 66 and 50 A(2) per heme for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. The trend of the m- and n(B)-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.
authors.affiliation: Department of Biological Sciences, Biochemistry Building, Imperial College of Science, Technology, and Medicine, London SW7 2AY, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fantuzzi
authors.forename: Sheila
authors.initials: S
authors.lastname: Sadeghi
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.forename: Gian Luigi
authors.initials: GL
authors.lastname: Rossi
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D016969:Desulfovibrio vulgaris / Q000201:enzymology; D019281:Dimerization; D005998:Glycine; D008715:Methionine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009565:Nitrate Reductases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D011994:Recombinant Proteins; C083951:cytochrome c553; C022302:cytochrome C-552; D008715:Methionine; D009565:Nitrate Reductases; D003545:Cysteine; D005998:Glycine