The structure of horseradish peroxidase C characterized as a molten globule state after Ca(2+) depletion.
The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.
Szigeti
Krisztián
K
Research Group for Membrane Biology, Hungarian Academy of Sciences, 1113 Budapest, Hungary.
Smeller
László
L
Osváth
Szabolcs
S
Majer
Zsuzsanna
Z
Fidy
Judit
J
D031215:Armoracia / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism / Q000379:methods; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D010940:Plant Proteins; D006735:Horseradish Peroxidase; D002118:Calcium