**7. References**


**11** 

*USA* 

**Time Resolved Thermodynamics Associated with Diatomic Ligand** 

*Department of Chemistry and Biochemistry, Florida International University Miami FL* 

Ligand-induced conformational transitions play an eminent role in the biological activity of proteins including recognition, signal transduction, and membrane trafficking. Conformational transitions occur over a broad time range starting from picosecond transitions that reflect reorientation of amino acid side chains to slower dynamics on the millisecond time-scale that correspond to larger domain reorganization (Henzler-Wildman et al., 2007). Direct characterization of the dynamics and energetics associated with conformational changes over such a broad time range remains challenging due to limitations in experimental protocols and often due to the absence of a suitable molecular probe through which to detect structural reorganization. Photothermal methods such as photoacoustic calorimetry (PAC) and photothermal beam deflection provide a unique approach to characterize conformational transitions in terms of time resolved volume and enthalpy changes (Gensch&Viappiani, 2003; Miksovska&Larsen, 2003). Unlike traditional spectroscopic techniques that are sensitive to structural changes confined to the vicinity of a chromophore, photothermal methods monitor overall changes in volume and enthalpy allowing for the detection of structural transitions that are spectroscopically silent (i.e. do

not lead to optical perturbations of either intrinsic or extrinsic chromophores).

Myoglobin (Mb) and hemoglobin (Hb) play a crucial role in the storage and transport of oxygen molecules in vertebrates and have served as model systems for understanding the mechanism through which protein dynamics regulate ligand access to the active site, ligand affinity and specificity, and, in the case of hemoglobin, oxygen binding cooperativity. Mb and individual α- and β- subunits of Hb exhibit significant structural similarities, i.e. the presence of a five coordinate heme iron with a His residue coordinated to the central iron (proximal ligand) and a characteristic "3-on-3" globin fold (Fig. 1)(Park et al., 2006; Yang&Phillips Jr, 1996). Both proteins reversibly bind small gaseous ligands such as O2, CO, and NO. The photo-cleavable Fe-ligand bond allows for the monitoring of transient deoxy intermediates using time-resolved absorption spectroscopy (Carver et al., 1990; Esquerra et al., 2010; Gibson et al., 1986) and time resolved X-ray crystallography (Milani et al., 2008; Šrajer et al., 2001). Based on spectroscopic data and molecular dynamics approaches (Bossa et al., 2004; Mouawad et al., 2005), a comprehensive molecular mechanism for ligand migration in Mb was proposed including an initial diffusion of the photo-dissociated CO molecule into the internal network of hydrophobic cavities, followed by a return

**1. Introduction** 

**Dissociation from Globins** 

Jaroslava Miksovska and Luisana Astudillo

