The specific aims of this proposal include: 1) the non-isothermal determination of the entropic and enthalpic contribution to the reduction potential (E(0') and stability of cytochrome c, using 18 different interrelated single and double mutants of rat cytochrome c. The key reaction evaluated for these cytochromes c is: Cyt c(ox) + e(-) --> Cyt c(Red); S(0')(Rxn) will be determined by measuring the E(0') versus temperature under non-isothermal conditions with direct square-wave voltammetry. The entropies of reaction reflect the reorganization of the protein going from the oxidized to the reduced state; 2) to use the known X-ray crystallographic structures of both the oxidized and reduced protein to interpret the extent to which specific changes in structure, e.g. hydrogen bonds either formed or eliminated via mutation, reflect themselves in changes of both S(0') and H(0'), and thus E(0'); 3) to test in a rigorous fashion with these cytochromes c the proposal that redox proteins undergo entropic/enthalpic compensation when the reduction potential is altered; 4) to test, in those cases where a second mutation compensates and corrects for the effects of the first mutation, if S(0') and H(0') are also returned to their original values and how S(0')Rxn changes are explained by structure; 5) to test the extent to which the entropy of reaction is related to the global stability and/or local stability of the protein, each of which has already been measured independently; 6) to apply Surface Enhanced Resonance Raman Spectroscopy (SERRS), to interrogate the molecular structure and orientation changes of adsorbed cytochrome c (especially the local vibration states of the iron heme) initiated by electrode potential or mutation, and to relate these spectroscopic changes to the S(0') and H(0') for each mutant; and 7) to determine the global stabilities of the wild type and mutant cytochromes in the reduced state, since nearly all previous work on global stability was with oxidized cytochromes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
7R15GM055892-02
Application #
6403278
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Ikeda, Richard A
Project Start
1997-05-01
Project End
2001-09-30
Budget Start
2000-07-01
Budget End
2001-09-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Wisconsin Milwaukee
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Milwaukee
State
WI
Country
United States
Zip Code
53201