www.acsprf.org

Artificial photosynthesis schemes, where the energy from sunlight is converted into chemical fuels, represent the largest untapped solution to the global energy crisis. To be viable, these schemes require a catalyst that can convert water into O₂. While most efforts developing these catalysts are synthetic, we decided to take advantage of an enzymatic catalyst that already accelerates the water-dioxygen interconversion and see if it can be harnessed for the desired reaction. We are extensively mutating the small laccase, SLAC, to couple it to electrodes and to enhance its reactivity.

Student Aharon Rosenbloom, based on our Y229K mutant with greatly enhanced activity, made a series of mutations introducing greater positive charge into the binding pocket of the enzyme. Despite the measured rates being an order of magnitude below the diffusion limit, none of these mutations showed enhanced reactivity higher than that of Y229K. Chemical oxidation of the enzyme at potentials necessary to split water (817 mV vs. SHE at pH 7) results in decomposition of the enzyme, suggesting that any high-powered oxidation chemistry performed by these enzymes will have to be driven by direct electrochemistry (where electron transfer can be finely controlled).

Undergraduate Dani Schoenfeld made several types of modified electrode surfaces in attempts to interface with SLAC. In one case, he generated the azo-dye derivative of aminoanthracene and coupled it to edge-plane graphite. Anthracene binds in the surface active sites of some laccases and so represented an attractive target. He also used EDC/NHS coupling to bind DOPA and tyrosine (which we know binds SLAC) to an electrode surface. In all cases, the surfaces were highly functionalized and active, but did not bind SLAC in a detectable manner. We believe that, based on the crystal structure, the substrates need to be linked to the electrode surface by a longer, flexible tether (which we are in the process of testing). Dani has also developed our protocol for spectroelectrochemistry and has synthesized metal-cyano derivatives that allow us to measure the reduction potentials of laccases over a four-hundred mV range.

Students Joseph Novetsky and Solly Silverman were successful in making three single mutants with elevated reduction potentials and began making mutants that combine two or three of these mutations. We are measuring the thermodynamics of reduction in the single mutants and will submit a paper on this topic shortly. Aharon Rosenbloom has taken these mutants and is measuring their rates of substrate-oxidation, oxygen-reduction, and intramolecular electron transfer rate. He is in the process of showing how these rates depend on the potentials in a systematic manner.

Finally, we have been making progress on modifying the trinuclear cluster of the enzyme. Our previous work showed that this site provides some level of stability for the enzyme trimer, since copper ligands are shared between monomers. We desired to increase the stability of the enzyme so that mutations at this critical site do not structurally cripple the enzyme. As such, undergraduate Chaim Szachtel introduced a disulfide bond (I262C/G264C) to bridge between monomers. The resulting enzyme maintains its trimeric structure, even after being boiled in SDS-PAGE buffer. This mutant is now the template for future mutations in the trinuclear cluster.

In summary, we have generated ten mutants of SLAC, including several with elevated reduction potentials. We have successfully developed spectroelectrochemistry and enzyme activity methodologies as a well as a platform for further mutations that exhibits extreme structural stability.