Probing the conformational mobility of the active site of a heme peroxidase.

We have previously demonstrated (Badyal et al., J. Biol.

Asymmetric mode of Ca²⁺-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling.

Filament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca²⁺-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca²⁺-dependent binding sites of each subunit in the dimer.

Proton delivery to ferryl heme in a heme peroxidase: enzymatic use of the Grotthuss mechanism.

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path.

Mechanism of the Ca²+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA.

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca(2+) binds to the EF2 domain of S100A4 with micromolar affinity and that the K(d) value for Ca(2+) is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K(d) results from a reduced dissociation rate constant (from 16 s(-1) to 0.

Evidence for heme oxygenase activity in a heme peroxidase.

The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase.

Iron oxidation state modulates active site structure in a heme peroxidase.

We have previously shown [Badyal, S. K., et al.

The redox properties of ascorbate peroxidase.

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials.

Autocatalytic formation of a covalent link between tryptophan 41 and the heme in ascorbate peroxidase.

Electronic spectroscopy, HPLC analyses, and mass spectrometry (MALDI-TOF and MS/MS) have been used to show that a covalent link from the heme to the distal Trp41 can occur on exposure of ascorbate peroxidase (APX) to H2O2 under noncatalytic conditions. Parallel analyses with the W41A variant and with APX reconstituted with deuteroheme clearly indicate that the covalent link does not form in the absence of either Trp41 or the heme vinyl groups. The presence of substrate also precludes formation of the link.

Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis.

Previous work [Sharp, K. H., et al.

Conformational mobility in the active site of a heme peroxidase.

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis.