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Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin
T C Pochapsky1, M Kostic, N Jain
1Departments of Chemistry and Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Biochemistry
|May 9, 2001
Summary
Putidaredoxin
Area of Science:
- Biochemistry
- Protein Dynamics
- Redox Biology
Background:
- Putidaredoxin (Pdx) is a ferredoxin involved in electron transfer.
- Pdx exhibits redox-dependent binding to cytochrome P450(cam) (CYP101).
- The reduced form of Pdx (Pdx(r)) binds more strongly to CYP101 than the oxidized form (Pdx(o)).
Purpose of the Study:
- To investigate the mechanism of redox-dependent conformational selection in Pdx.
- To elucidate the role of the metal cluster binding loop in Pdx dynamics.
- To provide direct evidence for redox-dependent conformational ensembles.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy (15N and 13C resonances).
- Paramagnetic relaxation effects.
- Site-directed mutagenesis (G40N mutation in Pdx).
Main Results:
- The metal cluster binding loop is the primary determinant of redox-dependent conformational changes in Pdx.
- Upon reduction, the metal cluster binding loop contracts, reducing Pdx dynamics.
- A G40N mutation allows direct observation of slow conformational exchange in Pdx(o).
- Pdx(r) populates fewer conformational substates than Pdx(o), confirming conformational selection.
Conclusions:
- Conformational selection within the Fe2S2 cluster binding loop drives redox-dependent protein dynamics in Pdx.
- These findings offer a mechanism for coupling Pdx oxidation state to CYP101 binding affinity.
- This study provides the first direct observation of slow-exchange, redox-dependent conformational ensembles.