Jose F. Cerda

Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal-tetrapyrrole cofactors, creating a 100 μs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids in protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.

Hydrogen bond-free flavin redox properties: managing flavins in extreme aprotic solvents

We report a simple, single step reaction that transforms riboflavin, which is insoluble in benzene, to tetraphenylacetyl riboflavin (TPARF), which is soluble in this solvent to over 250 mM. Electrochemical analysis of TPARF both alone and in complexes with two benzene-soluble flavin receptors: dibenzylamidopyridine (DBAP) and monobenzylamidopyridine (MBAP), prove that this model system behaves similarly to other previously studied flavin model systems which were soluble only in more polar solvents such as dichloromethane. Binding titrations in both benzene and dichloromethane show that the association constants of TPARF with its ligands are over an order of magnitude higher in benzene than dichloromethane, a consequence of the fact that benzene does not compete for H-bonds, but dichloromethane does. The alteration induced in the visible spectrum of TPARF in benzene upon the addition of DBAP is very similar to the difference produced by transfer to dichloromethane, further indicating that the flavin head group engages in a similar degree of hydrogen bonding with dichloromethane as with its ligands. This work underlines the importance of using a truly nonpolar solvent for the analysis of the effects of hydrogen bonding on the energetics of any biomimetic host-guest model system.

Electrochemical and structural coupling of the naphthoquinone amino acid.

As a prelude to engineering artificial energy conversion proteins emulating biology, we examine the inclusion of a synthetic naphthoquinone amino acid in a characterized host-guest protein and determine the effects of its quinone and hydroquinone forms on the helix-coil distribution.

A three-dimensional printed cell for rapid, low-volume spectroelectrochemistry.

We have used three-dimensional (3D) printing technology to create an inexpensive spectroelectrochemical cell insert that fits inside a standard cuvette and can be used with any transmission spectrometer. The cell positions the working, counter, and reference electrodes and has an interior volume of approximately 200 μl while simultaneously providing a full 1-cm path length for spectroscopic measurements. This method reduces the time required to perform a potentiometric titration on a molecule compared with standard chemical titration methods and achieves complete electrolysis of protein samples within minutes. Thus, the device combines the best aspects of thin-layer cells and standard potentiometry.

Evaluation of Heme Peripheral Group Interactions in Extremely Low-Dielectric Constant Media and Their Contributions to the Heme Reduction Potential.

In this study, we measured the contributions of the ionization of the heme propionates to the reduction potentials of heme b and heme a (bis)N-methylimidazole complexes in various low-dielectric constant conditions. Additionally, we measured the effects of H-bond to the heme a formyl group on the reduction potential of the heme. The performed electrochemical measurements show that ionization of the heme propionates lead to the largest redox change in dichloromethane with no electrolyte. The measured reduction potential changes for heme b and heme a were -55 and -47 mV (±10 mV) per ionized propionate, respectively. For heme a, the study demonstrates how the dielectric constant of the medium is important in the magnification of the ΔpKa upon redox-linked ionization of the heme propionates and their roles in the proton pump of cytochrome c oxidase.