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Year 1 Summary:

            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 at developing these catalysts are synthetic in nature, 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.

            Once equipment that could measure the concentration of O₂ was received, Dr. Machczynski tested both wild type SLAC and a commercially available laccase using chemical oxidants to provide driving force for the reaction. Under both pH 5 and 8, no oxygen production was detected. In order to improve the enzyme’s reactivity with negatively-charged oxidants, we introduced a positively-charged amino acid, Y229K, in the substrate binding pocket. While this mutation did not produce oxygen, when we tested it with typical laccase substrate Fe(CN)₆^4-, we found that increased binding was reflected in a decreased K_m but were surprised to see that k_cat was increased by two orders of magnitude despite little change in reduction potential. Undergraduate Aharon Rosenbloom has been constructing a family of mutants to address this phenomenon and we expect to publish on it in Year 2.

             Undergraduate student Rafi Huntley reproduced literature preps to create several types of stable self-assembled monolayers (SAMs) on the surface of Au electrodes and has been working to couple SLAC to the SAMs. Surface cysteine mutants, M296C and E228C, generated to form a disulfide bond between the enzyme and SAMs that had exposed thiols on the surface, as we had proposed, but we have not seen results yet from that approach. Aharon Rosenbloom developed new methodologies involving these Cys mutants, because several typical procedures would not work in the presence of the active enzyme. Some success has been achieved with an amino-terminated octanethiol, which provides a positively charged SAM surface. However, the coverage amounts to only ~10% and no catalytic current is observed. We also tried several other methods for protein film formation using edge-plane graphite, glassy carbon, and basal-plane graphite, but have seen no success with those techniques.

            Another major direction of the project is to increase the reduction potential of the Type-1 site. Undergraduate Joseph Novetsky constructed the mutants M298L and M298F, which are typical mutations at Type-1 sites that enhance the reduction potential by 100-250 mV while maintaining the other properties of the protein. In the case of SLAC, the normal blue protein was changed to purple and green in the case of the M298L and M298F mutations, respectively. The spectra resemble those of Type-1 sites that have had an additional carboxylic acid bind the copper ion. Accordingly, we began to convert the three nearby Asp and Glu residues into Thr in the M298L mutant. All of the mutants were purple and identical spectroscopically. We titrated cyanide as an exogenous ligand to bind Cu(II) and displace any such ligands and found that there was indeed an effect of the mutations. As acidic residues were removed, the K_d for cyanide binding decreased four orders of magnitude. Redox titrations and spectroelectrochemistry show that the reduction potentials are not elevated for these mutants. We are crystallizing these mutants to narrow down the issue, but are also making other mutations to increase the reduction potential of the Type-1 site, including increasing the hydrophobicity on the side of the Cu ion opposite the axial Met and tweaking the nearby hydrogen bonding network.  We have also recently purified the C288Y mutant of SLAC, which does away with the Type-1 Cu ion completely, replacing it with a Tyr residue which should have a reduction potential of 0.65 -0.85 V vs NHE at high pH, increasing as the pH is lowered.

            A third direction of the project is to introduce a photosystem II-like site in place of the Type-2 site in the trinuclear cluster of SLAC. Undergraduate Zachary Landy constructed the H102A/H234A mutant of SLAC and found that it decayed over the course of two weeks in the refrigerator. Zachary was also responsible for adapting several activity assays as well as our protocol for preparing samples for atomic absorption, which is atypical due to the stability of the enzyme. Since we would like to modify the trinuclear copper site (which includes the Type-2 site) and this site helps to hold the enzyme’s trimer together (it shares ligands with another protein chain), a new student will work on introducing a disulfide bond at the monomer-monomer interface to stabilize the trimer, but far away from the copper sites to avoid influencing activity. The only other Cys residue appears in the Type-1 site, so we don’t expect any interference.

            In summary, we have generated thirteen mutants of SLAC, including mutations that alter the fundamental properties of the enzyme. We have overcome the hurdles with basic characterization of such an active enzyme which gives us control over the quality of our samples. We continue to develop methods for electrochemical control of the enzyme and are generating further generations of mutants.