Benjamin A. Feinberg

Analytical Chemistry
Professor Emeritus
PhD, University of Kansas
(414) 229-4169
e-mail: feinberg@uwm.edu
Selected Publications

Our research interests focus on the structure, function, electron transfer mechanisms, redox properties, and reactivity of cytochromes c and iron-sulfur proteins and enzymes, and other redox active proteins. In addition to other biophysical approaches, we also make novel applications of electroanalytical chemistry to study these metalloproteins, including the electrochemical detection of short-lived intermediates.

In our most recent work we are pursuing both thermodynamic and kinetic studies of cytochrome c and its mutants. For example, we are making non-isothermal electrochemical measurements to determine the entropies of reaction for several mutants of cytochrome c. The key reaction for which the entropy of reaction, , is determined is: .

These values are determined by measuring the versus temperature under non-isothermal conditions with direct square-wave voltammetery and small volumes of protein. In the non-isothermal electrochemical experiments, the entropic () and enthalpic () contributions to the reduction potential () for both wild type and mutant proteins are being determined. These measurements will then be correlated to known structure and measured protein stability. We have developed a thermodynamic scheme that shows both how and to what extent these thermodynamic changes modulate protein stability.

From the viewpoint of dynamics, we have made a direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant. This work reveals the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetery showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in oxidized state the His82-Fe3+-His18 form predominates. The addition or removal of an electron to the appropriate form of this variant controls which coordination form predominates. Using the 2X2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms. These same changes in coordination have been shown to take place in the enzyme nitrite reductase during its catalytic cycle.

Thermodynamic parameters, including the entropy of reaction,, were determined for the net reduction/rearrangement reaction:

and compared to those for wild type cytochrome:

For the Phe82His variant mixed redox couple,

compared to

for the wild type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction. We are presently evaluating other Phe-82 mutants to determine in much more detail why the Phe82His mutant rearranges at all. Additionally, high speed cyclic voltammetric studies are in progress (up to 300 V/s) in order to obtain better simulations by "catching" up with the fastest rearrangement: