Spectroscopic markers of the T X R quaternary transition in human hemoglobin Giorgio Schiro `, Marco Cammarata, Matteo Levantino, Antonio Cupane * National Institute for the Physics of Matter (INFM) and Department of Physical and Astronomical Sciences, University of Palermo, via Archirafi 36, I-90123, Palermo, Italy Received 7 October 2004; received in revised form 4 November 2004; accepted 4 November 2004 Abstract In this work, we use a sol–gel protocol to trap and compare the R and T quaternary states of both the deoxygenated (deoxyHb) and carbonmonoxide (HbCO) derivatives of human hemoglobin. The near infrared optical absorption band III and the infrared CO stretching band are used to detect the effect of quaternary structure on the spectral properties of deoxyHb and HbCO; comparison with myoglobin allows for an assessment of tertiary and quaternary contributions to the measured band shifts. The RX T transition is shown to cause a blue shift of the band III by ~35 cm 1 for deoxyHb and a red shift of the CO stretching band by only ~0.3 cm 1 for HbCO. This clearly shows that quaternary structure changes are transmitted to the heme pocket and that effects on deoxyHb are much larger than on HbCO, at least as far as the band energies are concerned. Experiments performed in the ample temperature interval of 300–10K show that the above quaternary structure effects are bstaticQ and do not influence the dynamic properties of the heme pocket, at least as probed by the temperature dependence of band III and of the CO stretching band. The availability of quaternary structure sensitive spectroscopic markers and the quantitative measurement of the quaternary structure contribution to band shifts will be of considerable help in the analysis of flash-photolysis experiments on hemoglobin. Moreover, it will enable one to characterize the dynamic properties of functionally relevant hemoglobin intermediates and to study the kinetics of both the TYR and RYT quaternary transitions through time-resolved spectroscopy. D 2004 Elsevier B.V. All rights reserved. Keywords: FTIR spectroscopy; Band III; CO stretching band; Sol–gel encapsulation; Low temperature spectroscopy 1. Introduction Quaternary conformational changes are of utmost importance to protein function. The best known example is probably the TX R transition of hemoglobin, which is responsible for the cooperative ligand binding of this protein [1]. To monitor the conformational changes involved in hemoglobin function, it is mandatory to have experimentally accessible markers that not only depend on the structural details but also enable one to distinguish between spectral contributions arising from different levels of protein structure (i.e., tertiary vs. quaternary) and from its ligation state. Decoupling the protein quaternary conformation from ligation state is of particular relevance in the case of hemoglobin since it may help identifying and characterizing intermediate species (e.g., T-state liganded or R-state unliganded hemoglobin) that are relevant to understand the functional behavior of this protein. The search for quaternary structure sensitive spectroscopic markers has a long story. In the classical studies of Perutz et al. [2–5], ferric hemoglobin and/or mutant or chemically modified ferrous hemoglobins in the presence and absence of the allosteric effector inositol-hexaphosphate (IHP) were used to find quaternary structure sensitive spectroscopic markers. Iron–cobalt hybrid hemoglobins have also been investigated to study the effects of quaternary structure on various spectroscopic properties [6,7]. More recently, sol–gel encapsulation protocols [8–10] have been used to dramatically slow down the kinetics of 0301-4622/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2004.11.005 * Corresponding author. Tel.: +39 091 6234221; fax: +39 091 6162461. E-mail address: cupane@fisica.unipa.it (A. Cupane). Biophysical Chemistry 114 (2005) 27– 33 http://www.elsevier.com/locate/biophyschem