Concerted simulations reveal how peroxidase compound III formation results in cellular oscillations

Abstract

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.

FIGURE 1

Schematic representation of the elementary steps involving enzymatic intermediates in the peroxidase-oxidase reaction. Several additional nonenzymatic steps also take place. AH represents NADH or certain aromatic compounds such as melatonin. The enzyme can adopt one of five states: Per²⁺ and Per³⁺, and compounds (co) I to III with a bound ligand. The step shown by the thick arrow (binding of superoxide to form compound III) is simulated by Brownian dynamics to compute the second-order rate constant. The complete set of reactions is given, together with rate constants, in Table A1 of Appendix A.

FIGURE 2

Time series from biochemical network simulations showing oscillation of the concentrations of NAD(P)H and superoxide using the parameters given in Table A1. Initial concentrations of myeloperoxidase and melatonin were 200 and 300 μM, respectively. All other initial concentrations were zero. The bottom figure shows the envelope of the oscillations of NAD(P)H plotted against the second-order rate constant for the association of ferric myeloperoxidase and superoxide.

FIGURE 3

Superposition of the active sites of Cu/Zn SOD-B (red) and the MPO (yellow) and HRP (blue) heme proteins. A conserved water site is shown by a sphere in the SOD and MPO structures. A cyanide molecule is shown in the same location in the HRP structure where a recent crystal structure shows superoxide binds (Berglund et al., 2002). Despite lack of sequence similarity, there are similar features in the active sites, such as the location of the guanadinium group of an arginine. The distal histidines of MPO and HRP superimpose well, even though the entrance channels to the active sites of these two peroxidases have completely different shapes and orientations. The channel in MPO extends into the page behind and to the right of the distal histidine, whereas that in HRP extends out of the page in front of the distal histidine.

FIGURE 4

Electrostatic potential of the MPO homodimer at pH 5 (a) and pH 8 (b) contoured at + 0.5 (blue) and − 0.5 (red) kT/e. The black arrows show the locations of channels into the active sites. The yellow contours represent the reaction criterion for superoxide binding and are 8 Å from the Fe atom. The difference in the region of negative contours near the active sites at the two pHs is due to a histidine residue that changes protonation state between these pHs.

FIGURE 4

Electrostatic potential of the MPO homodimer at pH 5 (a) and pH 8 (b) contoured at + 0.5 (blue) and − 0.5 (red) kT/e. The black arrows show the locations of channels into the active sites. The yellow contours represent the reaction criterion for superoxide binding and are 8 Å from the Fe atom. The difference in the region of negative contours near the active sites at the two pHs is due to a histidine residue that changes protonation state between these pHs.