Heme Attachment Motif Mobility Tunes Cytochrome c Redox Potential

• Published in: Biochemistry (Easton) Vol. 46; no. 42; pp. 11753 - 11760
• Main Authors: Michel, Lea V, Ye, Tao, Bowman, Sarah E. J, Levin, Benjamin D, Hahn, Megan A, Russell, Brandy S, Elliott, Sean J, Bren, Kara L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.10.2007
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacteria - enzymology; Bacteria - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Cytochrome c Group - isolation & purification; Deuterium - chemistry; Electrochemistry; Genetic Variation; Heme - chemistry; Histidine - chemistry; Hydrogen - chemistry; Hydrogen Bonding; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutation; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Plasmids; Protein Conformation; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Solubility; Templates, Genetic
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701177j

Hydrogen exchange (HX) rates and midpoint potentials (E m) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c 551) and Hydrogenobacter thermophilus (Ht cyt c 552) have been characterized in an effort to develop an understanding of the impact of properties of the Cys-X-X-Cys-His pentapeptide c-heme attachment (CXXCH) motif on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c 552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c 551. Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations on HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as a decreased E m (by 81 mV), whereas the corresponding double mutant of Pa cyt c 551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in E m (by 30 mV). The changes in E m correlate with changes in axial His chemical shifts in the ferric proteins reflecting the extent of histidinate character. Thus, the mobility of the CXXCH pentapeptide is found to impact the His−Fe(III) interaction and therefore the heme redox potential.