The catalytic cycle of Fe(II)-containing Homoprotocatechuate 2,3-dioxygenase (HPCD) has been studied by mutating key active site amino acids such as His200 and by using various substrate analogs. The initial steps of the proposed catalytic cycle involve binding of homoprotocatechuate (HPCA: 3,4-dihydroxyphenylacetate) and O2 in adjacent ligand sites of the active site Fe(II), resulting in electron transfer from HPCA to O2 via the iron. The present study probes this process by introducing a His200 to cysteine (H200C) mutation. Spectroscopic evidence shows that an exceptionally long-lived oxygenated intermediate (H200CHPCAInt1) is formed that can be trapped in nearly 100% yield by rapid freeze quench procedures. By combining the results of crystallography, kinetic and optical studies, and M�ssbauer and EPR spectroscopies with Density Functional Theory (DFT) computations, we have gained insight into the mechanistic transformations that occur: (i) when HPCD binds HPCA and (ii) when O2 binds to the complex. The well-resolved M�ssbauer spectra of H200CHPCAInt1 show that the iron is inherently high-spin ferric (S = 5/2), but parallel-mode X-band EPR spectra show that the oxygen complex has an S = 2 ground state spin. Together, these findings demonstrate the presence of an exchange-coupled Fe(III)-radical pair (J > 10 cm-1). Spin-coupled systems such as this are of particular interest because they represent intermediates where radical character is localized either on the catechol ring or on a superoxo ligand, thereby allowing the mechanistically important shift in electron density to be studied in detail. Such shifts are believed to be critical in the reaction cycles of many enzymes from the large 2-His 1-Carboxylate (2H1C) family of nonheme iron enzymes. We have been able to characterize the H200CHPCAInt1 intermediate in detail, aiding us in developing models for understanding, at an orbital level, the process of substrate and O2 binding in HPCD and potentially other 2H1C enzymes.

Characterization of a new, long-lived intermediate in the His200Cys mutant system of homoprotocatechuate 2,3-dioxygenase by M�ssbauer, EPR, and DFT methods

248th National Meeting (2014)

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