Abstract
Hydrogen exchange (HX) rates and midpoint potentials (Em) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c551) and Hydrogenobacter thermophilus (Ht cyt c552) 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 c552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c551. 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 Em (by 81 mV), whereas the corresponding double mutant of Pa cyt c551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in Em (by 30 mV). The changes in Em 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.