Structural influence of calcium on the heme cavity of cationic peanut peroxidase as determined by 1H-NMR spectroscopy.
The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (LiP) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of LiP have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 1H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 1H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 microM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.
Barber
K R
KR
Department of Biochemistry, University of Western Ontario, London, Canada.
Rodríguez Marañón
M J
MJ
Shaw
G S
GS
Van Huystee
R B
RB
D010367:Arachis / Q000201:enzymology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002412:Cations; D006418:Heme / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009195:Peroxidase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation
D002412:Cations; D006418:Heme; D009195:Peroxidase; D002118:Calcium