NMR will be used to probe the origin of the spectroscopic differences between reduced vertebrate and chloroplast [2Fe-25] ferredoxin and the conformational change that reduction induces to the protein in vertebrate ferredoxins. Selective isotopic labeling will be used to assign the paramagnetically shifted resonances, which are a sensitive indicator of cluster geometry. Density functional calculations on simple models of the reduced [2Fe-2S] cluster will allow analysis of the nature of geometric differences between chloroplast and human ferredoxin by comparison with existing spectroscopic data. More sophisticated models can then be developed, which will be used to calculate paramagnetic shifts for comparison with experiment, thereby generating a model which is consistent with all the data. Global 13C and 15N labeling and standard 2- and 3-D NMR techniques, as well as methods which exploit the paramagnetism, will be used to obtain solution structural information of the diamagnetic region of the protein in both redox states, providing insight into the reduction-induced conformational change. This will be melded onto the active site model developed above, thus giving a complete picture of the reduced ferredoxin structure. Given the absence structural information on any reduced ferredoxin, and that the reduced and oxidized structures are clearly different, this is the only valid approach to solution structure determination. Mutagenesis studies will be used to probe the factors which dictate the reduced [2Fe-2S] cluster geometry. This work will provide needed insight into how reduction alters ferredoxin's affinity for its redox partners in the steroid hormone biosynthetic pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM020497-01
Application #
6136400
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Cassatt, James
Project Start
2000-07-01
Project End
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
1
Fiscal Year
2000
Total Cost
$30,916
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Machonkin, Timothy E; Westler, William M; Markley, John L (2004) Strategy for the study of paramagnetic proteins with slow electronic relaxation rates by nmr spectroscopy: application to oxidized human [2Fe-2S] ferredoxin. J Am Chem Soc 126:5413-26
Lin, I-Jin; Gebel, Erika B; Machonkin, Timothy E et al. (2003) Correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum rubredoxin. J Am Chem Soc 125:1464-5
Machonkin, Timothy E; Westler, William M; Markley, John L (2002) (13)C[(13)C] 2D NMR: a novel strategy for the study of paramagnetic proteins with slow electronic relaxation rates. J Am Chem Soc 124:3204-5