Redox Properties of Mesophilic and Hyperthermophilic Rubredoxins as a Function of Pressure and Temperature

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Abstract
The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 °C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 °C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 °C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 °C and 1 atm, −93 mV at 95 °C and 1 atm, and 44 mV at 25 °C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, ΔV° = −27 mL/mol, ΔS° = −36 cal K-1 mol-1, and ΔH° = −10 kcal/mol, and for P. furiosus, ΔV° = −31 mL/mol, ΔS° = −41 cal K-1 mol-1, and ΔH° = −13 kcal/mol) from its pressure− and temperature−reduction potential profiles. The thermodynamic parameters for electron transfer (ΔV°, ΔS°, and ΔH°) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (εprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.