Unfolding and pH studies on manganese peroxidase: role of heme and calcium on secondary structure stability.
The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.
Banci
Lucia
L
Magnetic Resonance Center CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy. banci@cerm.unifi.it
Bartalesi
Ilaria
I
Ciofi-Baffoni
Simone
S
Tien
Ming
M
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000187:drug effects; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
D001059:Apoproteins; D020035:Holoenzymes; D006418:Heme; D014508:Urea; D010544:Peroxidases; C051129:manganese peroxidase; D002118:Calcium; D004229:Dithiothreitol