Temperature, pH, and solvent isotope dependent properties of the active sites of resting-state and cyanide-ligated recombinant cytochrome c peroxidase (H52L) revealed by proton hyperfine resonance spectra.
Comparative proton NMR studies have been carried out on high-spin and low-spin forms of recombinant native cytochrome c peroxidase (rCcP) and its His52 --> Leu variant. Proton NMR spectra of rCcP(H52L) (high spin) and rCcP(H52L)CN (low spin) reveal the presence of multiple enzyme forms in solution, whereas only single enzyme forms are found in spectra of wild-type and recombinant wild-type CcP and CcPCN near neutral pH. The spectroscopic behaviors of these forms have been studied in detail when pH, temperature, and solvent isotope composition were varied. For resting-state rCcP(H52L) the comparatively large NMR line widths compromise resolution, but two specific enzyme forms were found. They were interconvertible on the basis of varying temperature. For rCcP(H52L)CN four magnetically distinct enzyme forms were identified by NMR. It was found that these forms dynamically interconvert with changing pH, temperature, and solvent isotope composition (percent D(2)O). These studies have identified the alkaline titration of His52 and essentially identical alkaline enzyme forms for natWTCcPCN and rCcP(H52L)CN. From this work we interpret an essential role of His52 in CcP function to be preservation of a single active site structure in addition to the critical role of general base catalysis.
Satterlee
James D
JD
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
Savenkova
Marina I
MI
Foshay
Miriam
M
Erman
James E
JE
D019943:Amino Acid Substitution; D001665:Binding Sites; D003486:Cyanides / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013696:Temperature
D003486:Cyanides; D011522:Protons; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D006418:Heme; D006639:Histidine; D003578:Cytochrome-c Peroxidase; D007930:Leucine