doi:10.1016/j.ccr.2006.08.018 
Copyright © 2006 Elsevier B.V. All rights reserved.
Review
Time resolved thermodynamics of ligand binding to heme proteins
Randy W. Larsena, 
, 
and Jaroslava Mikšovskáb
aDepartment of Chemistry, University of South Florida, 4202 E. Fowler Ave. SCA 400, Tampa, FL 33620, USA
bDepartment of Chemistry, Marshall University, One John Marshall Drive, Huntington, WV 25755, USA
Received 30 April 2006;
accepted 25 August 2006.
Available online 30 August 2006.
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Abstract
Understanding the thermodynamics of ligand binding in metallo-proteins is of critical importance in probing the underlying energetic basis for the reaction mechanism. However, many kinetic events occurring subsequent to ligand binding/release are on a time scale outside of that accessible using traditional calorimetric techniques thus making the construction of thermodynamic profiles for early kinetic events difficult. Photothermal methods have enjoyed considerable success in probing photo-triggered reactions on timescales ranging from 
ns to >ms. These techniques, including photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD), have been applied to obtain both molar volume and enthalpy changes for a wide range of biological processes including ligand binding to metallo-proteins. Here we review the progress made to date in utilizing photothermal methods (PAC and PBD) to probe the thermodynamics of small molecule binding to heme proteins ranging from simple globin-type proteins to the more complex heme/copper oxidases.
Keywords: Photothermal methods; Photoacoustic calorimetry; Photothermal beam deflection; Heme; FixL; Cytochrome c oxidase; Myoglobin; Transient spectroscopy
Abbreviations: 2-MeIm, 2-methylimidazole; BjFixL, Bradorhizobium japonicum FixL; Cbo, cytochrome bo3 from E. coli; CcO, cytochrome c oxidase from bovine heart; CD/MCD, circular dichroism/magnetic circular dichroism; COMVCcO, CO-mixed valence cytochrome c oxidase; CTAB, cetylmethylammonium bromide; ET, electron transfer; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; Fe(II)4SP, FeII meso-tetrakis(4-sulphonatophenyl)-porphyrin; FeIIPPIX, Fe(II) protoporphyrin IX; FTIR, Fourier transform infra-red; HbA, human hemoglobin A; hhMB, horse heart myoglobin; HOMO/LUMO, highest occupied molecular orbital/lowest unoccupied molecular orbital; Mb, myoglobin; MP-11, microperoxidase-11; PAC, photoacoustic calorimetry; PBD, photothermal beam deflection; RbCcO, Rb. sphaeroides cytochrome c oxidase; SmFixL, Sinorhizobium meliloti FixL; swSMb, sperm whale myoglobin
Fig. 1. Structural diagram of both free-base and metallo-porphyrin illustrating the difference in porphyrin symmetry.
Fig. 2. Molecular orbital diagram (left) and molecular states for free-base porphyrin with D2h symmetry.
Fig. 3. Diagram showing the four orbital model used to describe the optical spectrum of metallo-porphyrins with D4h symmetry.
Fig. 4. Molecular orbital diagram for the porphyrin π-system (top) and transition metal atomic orbitals (bottom).
Fig. 5. Ribbon presentation of the structures of several classes of heme proteins. α-Helices are shown in red, β-sheets in blue and turn and random coil elements in gray.
Fig. 6. Diagram showing volume and enthalpy profiles that can be obtained utilizing time-resolved photothermal methods.
Fig. 7. Schematic diagram of both photoacoustic calorimtery and photothermal beam deflection instruments. Details are discussed in the text.
Fig. 8. Schematic presentation of heme model complexes: (H2O)FeII4SP is shown in (Panel A); (2-MeIm)FeII4SP in (Panel B); FeIIPPIX in a micelle is depicted in (Panel C); MP-11 is shown (Panel D).
Fig. 9. Typical photoacoustic trace for CO photodissociation from (CO)(2-MeIm)FeII4SP and reference (FeIII4SP in 50 mM Tris buffer pH 7) at 22 °C. The sample is shown as a solid line and the reference as dashed line. The absorbance of sample and the reference was 0.35 ± 0.02 at 532 nm.
Fig. 10. Plot of S/R Ehν vs. (Cpρβ) for CO photodissociation from (CO)FeII4SP, (CO)(H2O)FeII4SP and (CO)FeIIMP-11 complexes.
Fig. 11. PBD trace for CO photodissociation from (CO)(2-MeIm)FeII4SP and the reference compound (FeIII4SP). The sample trace is shown in black and the reference trace in gray.
Fig. 12. The plot of αi/REhν vs. Cpρ/(dn/dt) for CO photodissociation from and rebinding to (H2O)FeII4SP (circles), (2-MeIm)FeII4SP (squares), and FeIIMP-11 (triangles). αi for the fast phase is shown as open symbols and αi for the slow phase is shown as solid symbols.
Fig. 13. The overlay of PBD trace for CO rebinding to deoxyMb (dark line) and for the reference compound (gray trace) measured at 18 °C.
Fig. 14. The plot of αiEhν vs. (Cpρ)/(dn/dt) for the fast and slow phase (squares and circles, respectively).
Fig. 15. Ribbon diagram of the FixL heme domain X-ray crystal structure in the ligand free-state (PDB entry 1XJ4). The heme cofactor is shown together with Arg206 and Arg220.
Fig. 16. Photoacoustic trace for CO photodissociation from CO BjFixLH140–270 (Panel A) and BjFixLH151–256 (Panel B) at 35 °C including corresponding reference traces. Conditions: 10 μM protein, 50 mM Tris buffer, pH 7.5, 50 mM NaCl and 1 mM CO.
Fig. 17. Plot of 
iEhν vs. Cpρ/β for the fast phase (solid circle) and slow phase (open circle) of CO photodissociation from BjFixLH140–270 and for the single fast phase (solid square) from BjFixLH151–256.
Fig. 18. Top metal center orientation within bovine heart CcO. Bottom: Proposed catalytic mechanism for CcO.
Fig. 19. Thermodynamic profiles for the photolysis of CO from the fully reduced CO bound forms of bovine heart CcO (solid lines), E. coli cytochrome bo3 (dashed lines) and Rb CcO (dotted lines). The ‘boxed’ species represents the 500 ns transient, only observed for CORbCcO photolysis.
Fig. 20. Overlay of COMVCcO and calorimetric reference acoustic signals obtained at pH 6 (top left), pH 8 (top right) and pH 9 (bottom). Sample conditions: [CcO] = 16 μM in 50 mM HEPES buffer containing 0.05% β-d-laurylmaltoside.
Fig. 21. Deconvolution of the acoustic waves for COMVCcO obtained at various pHs. Plots show an overlay of acoustic waves for COMVCcO, calorimetric reference, three-exponential fit and residuals. Top panel: fits for pH 6; bottom panel: fits for pH 8.
Fig. 22. Plot of Ehν vs. Cpρ/β or the acoustic signals obtained at pH extremes, pH 6 and 9. The normalized amplitudes (ι) for the various phases at pH 6 were obtained from the deconvolution of the acoustic waves as per Fig. 21.
Fig. 23. Thermodynamic profiles (molar volume and enthalpy) for photolysis of COMVCcO at pHs < 9.
Table 1.
Volume and enthalpy changes for CO dissociation from heme model complexes determined by PAC
Table 2.
Reaction and activation volume and enthalpy changes for CO photodissociation (ΔVfast and ΔHfast) and rebinding (ΔVfast, ΔHfast and ΔV‡) to heme model complexes
Reaction parameters were determined using PBD whereas activation volume changes were determined from pressure dependence of rate constant for CO rebinding.
Table 3.
Thermodynamic parameters for CO photodissociation and rebinding to hhMb determined by PBD
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