Elucidating thermodynamic parameters for electron transfer proteins using isothermal titration calorimetry: application to the nitrogenase Fe protein

• Published in: Journal of biological inorganic chemistry 2003-05, Vol.8 (5), p.560-566
• Main Authors: Sørlie, Morten, Chan, Jeannine M, Wang, Haijang, Seefeldt, Lance C, Parker, Vernon D
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Triphosphate - chemistry; Calorimetry; Electron Transport; Nitrogenase - chemistry; Nonheme Iron Proteins - chemistry; Thermodynamics
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-003-0446-7

Establishing thermodynamic parameters for electron transfer reactions involving redox proteins is essential for a complete description of these important reactions. While various methods have been developed for measuring the Gibbs free energy change (Delta G(HR) or E(m)) for the protein half-reactions, deconvolution of the respective contributions of enthalpy (Delta H(HR)) and entropy (Delta S(HR)) changes is much more challenging. In the present work, an approach is developed using isothermal titration calorimetry (ITC) that allows accurate determination of all of these thermodynamic parameters for protein electron transfer half-reactions. The approach was validated for essentially irreversible and reversible electron transfer reactions between well-characterized mediators and between mediators and the protein cytochrome c. In all cases, the measured thermodynamic parameters were in excellent agreement with parameters determined by electrochemical methods. Finally, the calorimetry approach was used to determine thermodynamic parameters for electron transfer reactions of the nitrogenase Fe protein [4Fe-4S](2+/+) couple in the absence or presence of MgADP or MgATP. The E(m) value was found to change from -290 mV in the absence of nucleotides to -381 mV with MgATP and -423 mV with MgADP, consistent with earlier values. For the first time, the enthalpy (Delta H(HR)) and entropy (Delta S(HR)) contributions for each case were established, revealing shifts in the contribution of each thermodynamic parameter induced by nucleotide binding. The results are discussed in the context of current models for electron transfer in nitrogenase.