Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.
The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.
Wever
R
R
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
Oertling
W A
WA
Hoogland
H
H
Bolscher
B G
BG
Kim
Y
Y
Babcock
G T
GT
D002713:Chlorine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D009195:Peroxidase / Q000737:chemistry*; D011489:Protein Denaturation; D013059:Spectrum Analysis, Raman
D006146:Guanidines; D002713:Chlorine; D009195:Peroxidase; D019791:Guanidine