Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome.

The stability of human flavoproteins strongly depends on flavin levels, although the structural and energetic basis of this relationship is poorly understood. Here, we report an in-depth analysis on the thermodynamics of FAD binding to one of the most representative examples of such relationship, NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a dimeric enzyme that tightly binds FAD, which triggers large structural changes upon binding. A common cancer-associated polymorphism (P187S) severely compromises FAD binding. We show that FAD binding is described well by a thermodynamic model explicitly incorporating binding cooperativity when applied to different sets of calorimetric analyses and NQO1 variants, thus providing insight on the effects in vitro and in cells of cancer-associated P187S, its suppressor mutation H80R and the role of NQO1 C-terminal domain to modulate binding cooperativity and energetics. Furthermore, we show that FAD binding to NQO1 is very sensitive to physiologically relevant environmental conditions, such as the presence of phosphate buffer and salts. Overall, our results contribute to understanding at the molecular level the link between NQO1 stability and fluctuations of FAD levels intracellularly, and supports the notion that FAD binding energetics and cooperativity are fundamentally linked with the dynamic nature of apo-NQO1 conformational ensemble.

Iron binding effects on the kinetic stability and unfolding energetics of a thermophilic phenylalanine hydroxylase from Chloroflexus aurantiacus.

The effects of non-heme iron binding on the function, structure, and stability of a monomeric phenylalanine hydroxylase from the thermophile Chloroflexus aurantiacus (caPAH) were investigated. Comparative studies on holo (iron-bound) and apo (iron-depleted) caPAH indicated that iron(II) binding does not significantly affect the overall structure of the enzyme. Thermal denaturation studies performed using differential scanning calorimetry showed that the unfolding reaction was kinetically controlled and that holo-caPAH displayed a large increase in thermal stability (approximately 15 degrees C upshift in the T (m) value) compared with the apoenzyme. Analysis using a simple irreversible two-state model also showed a higher kinetic stability for holo-caPAH at optimal growth temperature (denaturing approximately 8 times more slowly than the apo form at 55 degrees C). Experiments performed in the presence of urea in combination with structure-energetics calculations suggest that iron binding reduces the change in accessible surface area exposed in the unfolding transition state (from approximately 36% to approximately 5% of the total change in accessible surface area) and also the surface involved in water-unsatisfied broken internal contacts (solvation barriers). Additional comparative analyses using phenylalanine hydroxylase from mesophilic and psychrophilic organisms suggest that, in addition to its catalytic role, the non-heme iron serves to enhance the kinetic stability of phenylalanine hydroxylase at the optimal growth temperature of the organism.

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.