Kovacs Group Projects

Our research program is aimed at determining how cysteinates influence function in non-heme iron enzymes. Non-heme iron enzymes promote a number of important biological reactions, including serotonin, leukotreine, and DNA synthesis. Cysteinate-ligated non-heme iron enzymes Superoxide reductase (SOR) and nitrile hydratase (NHase) are involved in the detoxification of superoxide (O2-) radicals, and nitrile wastes, respectively. Superoxide has been implicated in a number of disease states, including cancer, Alzheimers, and Parkinsons. The SOR active site closely resembles that of the heme enzyme cytochrome P450 (which oxidizes unactivated hydrocarbons), and both involve an Fe(III)-OOH intermediate. NHase belongs to a growing class of post-translationaly modified enzymes containing cysteine sulfenic acids. Neither the mechanism of O2- reduction by SOR, nor the function of sulfenic acid in NHase are well-understood.

In metalloenzymes, details regarding key bond lengths, the presence or absence of protons, or the identity of intermediates, can be "fuzzy" or lacking, due to limitations imposed by either the biological system, or physical technique. Quite often these details can be revealed via synthetic models. Only recently have synthetic Fe(III) -peroxo and Fe(IV)-oxo species been spectroscopcially characterized, and most of these contain nitrogen ligands. Since it is not clear that the correlation between peroxide binding mode, spin-state, and vibrational parameters, established for nitrogen-ligated Fe(III)-peroxos, would hold for thiolate-ligated Fe(III)-peroxos, there is a need for the latter, in order to provide benchmark parameters.

These questions, and more, are being explored in the Kovacs group by designing organic molecules that contain nitrogen and sulfur heteroatoms, and possess a molecular architecture that enforces a desired stereochemistry around a given metal ion. We then examine the reactivity of the resulting synthetic model complexes, and attempt to correlate this with structure and properties, such as spin–state and electronic structure, by systematically altering the structure of our organic ligands. Reactivity of these models is then compared on the basis of kinetic and thermodynamic parameters. Reactivity is monitored by low temperature electronic absorption spectroscopy, and by EPR. Kinetics data is obtained using electronic absorption spectroscopy, stopped–flow techniques, and NMR line–shape analysis.

SOR Models. We recently synthesized the first and only functional model for SOR, characterized the first reported example of a thiolate-ligated Fe(III)-peroxide, and were the first to report metrical parameters for a mononuclear Fe(III)-OOH in any ligand environment. Five-coordinate thiolate-ligated [Fe(II)(SMe2N4(tren))]+ (1) reduces O2- to afford H2O2 via a metastable peroxo intermediate, [Fe(III)(SMe2N4(tren))(OOH)]+ (2). This occurs via the oxidative addition of superoxide to 1, in a proton-dependent mechanism. No reaction occurs in the absence of a proton source. Hydrogen peroxide is released from # at a rate of 65(1) s-1 (at 298 K), comparable to the rate of H2O2 release in the SOR enzyme, affording a stable solvent-bound species [FeIII(SMe2N4(tren)(solv)]2+ (solv= MeCN, MeOH). The rate at which hydrogen peroxide is release is dependent upon the pKa of the proton donor. Acetic acid releases H2O2 to afford an acetate-bound derivative similar to the glutamate-bound form of SOR. To mimic the reduction of the oxidized FeIII state of SOR, and complete the catalytic cycle, cobaltacene was added as a source of electrons to the oxidized solvent-bound species #. This resulted in the regeneration of reduced [FeII(SMe2N4(tren))]+ (3), which subsequently reacts with additional superoxide to regenerate the peroxide intermediate (Figure 8). Eight turnovers have been achieved in this stepwise manner. We are currently examining the kinetics of both peroxo # formation, and H2O2 release in order to elucidate the mechanism.

NHase analogues. Prior to the X-ray crystal structure of NHase, our group showed that when two aliph-atic thiolates and two imines are incorporated into the coordination sphere, the magnetic (S= 1/2; g= 2.19, 2.13, 2.01) and electronic properties (intense green color; lmax= 718(1400) nm) of the resulting model compound, [Fe(III)(ADIT)2]+ (#), are remarkably similar to those of the NHase enzyme. The low–spin state of this model is unusual given that the ligand–field of p–donor ligands (e.g., SR–) tend to shrink 10Dq and thus favor a high–spin state. In collaboration with Solomon, we’ve looked into the structural features responsible for these electronic and magnetic properties, as well as those of NHase.

Nitrile hydratase is one of a growing number of enzymes shown to be post-translationaly modified, and contain cysteine sulfenic acids (RS-OH). The function, mechanism of formation, and protonation state of the singly oxygenated cysteine (RS=O) of NHase are unknown. It has been proposed that it is intimately involved in the mechanism.