The Importance of Bound Ca ions in Horseradish Peroxidase Function

Krisztian Szigeti, Monique Laberge, Judit Fidy

Semmelweis University Budapest, Department for Biophysics and Radiation Biology, MTA SE Research Group for Biophysics Hungarian Academy of Science

Native HRPC binds two Ca ions. The two Ca-binding sites were studied by experimental and computational methods. The essential structural elements include a 5-coordination protoporphyrin IX (b type Heme) with an extensive hydrogen bonding network and two Ca, one distal and one proximal to the heme. Molecular modeling studies show that the proximal site is partially solvent-exposed being in the more flexible region of the protein while the distal site is buried in the protein core. It was show that the depletion of Ca from the enzyme causes a change in the enzymatic activity of the protein, and some studies reported also conformational changes due to the removal of Ca. The literature data however were not coherent enough concerning the success of Ca depletion. We elaborated a protocol to reliability remove both Ca ions and investigated the conformational effect around the heme. By molecular modeling we unraveled that the distortion of the heme from planarity is required for function. The distortion is less pronounced after Ca-removal. We show experimental evidence be low temperature absorption spectroscopy and studying pH dependence by CD, Trp fluorescence and absorption spectroscopy that the conformation of the heme pocket is significantly influenced by the binding of Ca ions. Substrate binding studies unravel the conformational effect in the substrate-chanel. A structural model is suggest based on molecular dynamics simulation of the effect.

Email: sziget@puskin.sote.hu

Address: Semmelweis University, Department of Biophysics and Radiation Biology, Hungary, 1444 Budapest Pf 263