Research Interests of Edmond F. Bowden

Our major research focus is the electrochemistry of proteins and enzymes. Over the past decade, my group has been developing a strategy we refer to as “protein monolayer electrochemistry”, in which voltammetry experiments are conducted on a single molecular layer of a functional, surface-attached, redox protein or enzyme. This research area is quite interdisciplinary in nature, as you might imagine, and we operate with two major objectives in mind. First, from a biophysical/bioinorganic perspective, we are seeking to develop new insight into the kinetics and mechanisms of biological electron transfer reactions of metalloproteins through the application of modern electrochemical strategies and methods. Second, from a bioanalytical perspective, we are learning how to realize direct electronic coupling between electrodes and enzyme catalysts as a basis for developing electrochemical biosensors.

A critical aspect of our work is the development of biocompatible electrode surfaces. By controlling the molecular structure at the interface, we can immobilize single monolayers of a given protein in a fully functional and electroactive state. Organothiolate/gold modification chemistry has proven to be exceedingly useful for this task. In collaboration with synthetic chemists at NCSU, we prepare substituted alkanethiols, R(CH2)nSH (where n = 1 thru 20, and R = COOH, OH, NH2, CH3, etc.), which spontaneously react with gold surfaces via the thiol moiety to form compact, highly ordered and stable overlayers that are one molecule thick. These structures, referred to as self-assembled monolayers (SAMs), are currently the focus of many research efforts worldwide. The surface chemistry of SAMs, i.e., charge, acid-base properties, hydrophobicity, etc., can be tailored for interacting with a particular protein by variation of the R group substituent. More refined control of surface chemistry can be realized by preparing “mixed” SAMs containing 2 or more different alkanethiols. The polymethylene chain length (n) of a SAM can be varied to alter the electron tunneling distance between the active site in the protein and the gold substrate, and this provides us with a powerful means for controlling the kinetic timeframe of a given experiment. Currently we are utilizing a wide variety of substituted alkanethiols in our research program. Physical characterization of the SAM/gold structures is being extensively pursued using electrochemical and impedance methods, scanning probe microscopy (STM, AFM), and spectroscopic methods (UV/Vis electroreflectance spectroscopy, FTIR).

Which proteins are we working with? Cytochromes are truly excellent electron transfer proteins for electrochemical and spectroscopic studies and they continue to be one of our favorite molecules. The figure below shows one example from our work, namely, horse cytochrome c adsorbed to a COOH-terminated SAM-modified gold electrode. This SAM has been prepared from 16-mercaptohexadecanoic acid, which results in an anionic electrode surface that successfully binds the positively charged cytochrome c molecule. Cytochrome projects currently underway in our group include the electrochemistry of yeast cytochrome c and its genetically engineered variants, the electrochemistry of cytochrome b5 (an anionic protein that binds to cationic surfaces), electrochemistry of metal-substituted cytochrome c, determination of heme orientation in adsorbed cytochrome c using polarized UV/Vis electroreflectance spectroscopy, and STM/AFM imaging of cytochrome monolayers.

We are also interested in the fundamental molecular interpretation of the electron transfer rates that are electrochemically measured in the cytochrome/SAM/gold systems. Along these lines, we have developed linear-sweep voltammetric (LSV) theory that incorporates Marcus Theory kinetics and allows key parameters such as the reorganization energy to be directly extracted from voltammograms. We have also established the distance dependence of electron transfer by varying the SAM chain length, with results that are consistent with electron tunneling through a saturated hydrocarbon chain. Of great immediate interest is our attempt to unravel the specific bonded molecular pathways that are utilized for electron transfer across the SAM/cytochrome interface. Genetically engineered yeast cytochrome c mutants with single-site amino acid substitutions are being employed to address this objective.

Although much of our current work is focused on the cytochromes, we also remain very interested in the coupling of enzymes to electrodes. We have published several papers over recent years on the electrocatalytic behavior of cytochrome c peroxidase (CCP) adsorbed to edge-oriented pyrolytic graphite electrodes. Our current plans are to immobilize CCP on SAM/gold electrodes and to realize direct electronic coupling and efficient electrocatalysis. The cytochrome b5 project mentioned above actually provides the groundwork for pursuing these experiments since cytochrome b5 and the much more complicated CCP are both anionic proteins. The SAM/gold electrode will provide a much improved opportunity over graphite electrodes for obtaining molecular-level insight into the interfacial CCP electron transfer process.