High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution, ligand binding, Ca++ removal, and reduction of the disulfide bonds.
The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.
Smeller
L
L
Department of Biophysics & Radiation Biology, Semmelweis University, Puskin u. 9. PF 263, H-1444 Budapest, Hungary.
Meersman
F
F
Fidy
J
J
Heremans
K
K
D000577:Amides; D001051:Apoenzymes / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006877:Hydroxamic Acids / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000737:chemistry; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D000577:Amides; D001051:Apoenzymes; D004220:Disulfides; D006877:Hydroxamic Acids; D008024:Ligands; D008670:Metals; D011166:Porphyrins; D006418:Heme; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid; D002118:Calcium