Protein matrix local fluctuations and substrate binding in HRPC: A proposed dynamic electrostatic sampling method

Abstract

Horseradish peroxidase C is an oxidoreductase which catalyzes in plant roots the oxidation of a remarkably wide variety of aromatic compounds by H 2 O 2 . The recently available X-ray structures of the enzyme bound to aromatic substrates are not indicative of significant structural rearrangements as a result of substrate binding when compared to the structure of the unbound enzyme. Our most recent spectral hole-burning studies on HRPC fluorescent derivatives provided direct experimental evidence indicative of different fields experienced at the heme as a result of substrate binding which can only originate in the protein matrix in which the heme is embedded. In this report, we present results on modeling the fluctuations of these protein matrix electrostatic rearrangements using a combination of molecular dynamics and electrostatic conformational sampling to compare the binding of BHA and NHA to HRPC and to the derivative used in our experimental studies. Our initial results suggest that HRPC does undergo conformational rearrangement on binding aromatic substrates, but of an electrostatic nature, as opposed to major conformational structural changes. This implies that the high versatility of substrate binding in horseradish peroxidase could be due to an electrostatic dynamic selectivity mediated by small-amplitude internal protein matrix motions.

The Enzyme Horseradish Peroxidase Is Less Compressible at Higher Pressures

“…7 schematically shows this model. This interpretation is supported by results of recent molecular dynamics simulations on HRP in our laboratory (Schay et al, 2001) that showed internal cavities evolving around the heme site, and increased fluctuations of the electric potential around the active center of the enzyme. This pressure-tuned elasticity of the protein regions explains the difference between the results of the present measurements performed in the highpressure range, 0.1-1.7 GPa, and the hole-burning measurements in the low-pressure range, 0.1-1.1 MPa.…”

The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

“…Charges for the ferric heme and the Ca 21 -coordination spheres were calculated at the Hartree-Fock level using a 6-311G basis set and electrostatic potential fitting as previously described (Schay et al, 2001) and incorporated into CHARMM. van der Waals parameters were from Cates et al (2002).…”

“…Explicit hydrogens were added using the HBUILD module and the amino acid residues were protonated so as to be consistent with neutral pH. The propionic acid side chains were also considered ionized as discussed elsewhere (Schay et al, 2001) and the disulfide bridges were explicitly modeled, namely: Cys11-Cys91, Cys44-Cys49, Cys97-Cys301, and Cys177-Cys209. The structures were solvated in a 36-Å sphere of 5000 explicit TIP3 waters (Jorgensen et al, 1983) using a spherical shape quartic boundary potential within the Miscellaneous Mean Field Potential approximation as implemented in CHARMM.…”

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 structure of HRP in its complexed form with benzohydroxamic acid (BHA) (16) and with ferrulic acid (17) as substrates is also known. These results show that the major structural elements in the protein are R-helices (15). Additionally there are two Ca 2+ ions bound to the protein (18,19).…”

“…Its crystal structure was recently determined by Gajhede et al (14) and further refined by computer modeling (15). The structure of HRP in its complexed form with benzohydroxamic acid (BHA) (16) and with ferrulic acid (17) as substrates is also known.…”