Generation and Characterization of SAM Immobilized Enzyme Films in Ionic Liquids

Electrochemical investigations of the heme protein dehaloperoxidase (DHP) were undertaken. The motivation for this work lies in the unusual bifunctional nature of DHP, a globin-type protein found in the marine worm Amphitrite ornata. DHP is a monomeric hemoglobin that not only binds and transports dioxygen but that features a high level of peroxidase activity for the enzymatic detoxification of halogenated phenolic compounds such as 2,4,6-tribromophenol. Direct electrochemistry of DHP in the absence of mediators was realized for both diffusional and non-diffusional voltammetric cases using mixed OH/COOH self-assembled monolayer modified electrodes of alkanethiolate/gold construction. It appears that DHP interacts with anionic electrodes via a lysine patch situated in the vicinity of the heme edge. The interfacial voltammetry, however, suffers from instability problems that have not been completely resolved. Accordingly, mediated thin-layer spectroelectrochemistry (rather than cyclic voltammetry) was utilized to characterize the Fe(III)/Fe(II) redox thermodynamic properties of DHP. The reduction potential at pH 7 was determined under anaerobic conditions to be +202 mV vs SCE, the most positive value for any known intracellular globin. This unusual value was rationalized in terms of a redox-coupled conformational behavior involving the distal histidine (H55) by analyzing the Gibbs free energy contributions to the potential. The impact of enzyme substrate interactions on redox properties was also characterized and gave evidence for both internal and external binding depending upon the extent of halogen substitution of the phenolic substrate. A cubic thermodynamic cycle was proposed that accounted for both conformational changes and substrate interactions. Finally, several methionine-86 mutants of DHP were prepared to investigate the electron push effect concept for peroxidase activity. The first successful installation of a peroxide-like aspartate-histidine-iron triad into a globin protein was achieved (M86D mutant). (a) Papers published in peer-reviewed journals (N/A for none) J.D. D’Antonio, E.L. D’Antonio, M.K. Thompson, E.F. Bowden, T. Smirnova, S. Franzen, and R.A. Ghiladi, “Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B from Amphrite ornata”, Biochemistry, 2010, 49, 6600-6616. V.S. de Serrano, M.F. Davis, J.F. Gaff, Qi Zhang, Z. Chen, E.L. D’Antonio, E.F. Bowden, R. Rose, and S. Franzen, “X-ray structure of the metcyano form of dehaloperoxidase from Amphitrite ornata: evidence for photoreductive dissociation of the iron-cyanide bond”, Acta Crystallogr. D, 2010, 66, 770-782. List of papers submitted or published that acknowledge ARO support during this reporting period. List the papers, including journal references, in the following categories: (b) Papers published in non-peer-reviewed journals or in conference proceedings (N/A for none) 2.00 Number of Papers published in peer-reviewed journals: Number of Papers published in non peer-reviewed journals: (c) Presentations 0.00 Number of Presentations: 0.00 Non Peer-Reviewed Conference Proceeding publications (other than abstracts): Number of Non Peer-Reviewed Conference Proceeding publications (other than abstracts): 0 Peer-Reviewed Conference Proceeding publications (other than abstracts): (d) Manuscripts Number of Peer-Reviewed Conference Proceeding publications (other than abstracts): 0 Number of Manuscripts: 0.00