Ligand-Induced Tertiary Relaxations During the T-to-R Quaternary Transition in Hemoglobin Luca Ronda, † Stefania Abbruzzetti, ‡ Stefano Bruno, † Stefano Bettati, † Andrea Mozzarelli, † and Cristiano Viappiani* ,‡ Dipartimento di Biochimica e Biologia Molecolare, UniVersita ` degli Studi di Parma, CNISM, and Dipartimento di Fisica, UniVersita ` degli Studi di Parma, CNISM, and NEST CNR-INFM ReceiVed: April 8, 2008; ReVised Manuscript ReceiVed: June 24, 2008 When human hemoglobin is encapsulated in nanoporous silica gels, tertiary and quaternary structural relaxations are dramatically slowed down, allowing the characterization of elusive reaction intermediates. In this work, the conformational and functional changes triggered by CO binding to human deoxyhemoglobin gels were determined in the absence and presence of allosteric effectors. CO rebinding kinetics to human deoxyhemo- globin gels after nanosecond laser photolysis were monitored as a function of time after CO saturation. A maximum entropy analysis of the CO rebinding kinetics shows that the T conformation slowly evolves toward R, with an associated redistribution of tertiary species. The tertiary species are characterized by different CO rebinding rates which are essentially unaffected by the protein quaternary state. Introduction Binding of oxygen to hemoglobin (Hb) triggers tertiary conformational changes that culminate in the quaternary transi- tion from the deoxy (tense, T) to the oxy (relaxed, R) state. The time overlap of these structural transitions prevents the decoupling of the purely tertiary and quaternary changes in solution experiments where the kinetics of ligand binding is initiated by rapid mixing or laser flash photolysis of carbon monoxide (CO) complexes. The kinetic trace of CO rebinding after laser flash photolysis has been interpreted as originating from (i) the nanosecond geminate rebinding of CO still present in the heme pocket of R state Hb molecules that have not yet relaxed to the T state, (ii) the microsecond bimolecular binding of CO from the solvent to an ensemble of R state conformations, including relaxed, partially relaxed or unrelaxed subunits, and (iii) the millisecond bimolecular binding of CO from the solvent to subunits belonging to tetramers that have meanwhile switched to the T quaternary state. 1,2 Several strategies have been adopted to investigate these events separately, including the substitution of the heme Fe in some of the subunits with other metals, such as Ni, Co, and Zn (which favor the formation of half-liganded derivatives having the low-affinity T quaternary state conforma- tion 3-8 ), and the increase of solvent viscosity to selectively decrease the rate of one transition with respect to the others. 9 A recently applied powerful strategy consists of the encapsula- tion of proteins in silica gels. This approach allows us to trap defined quaternary states, slowing the rate of conformational transitions by several orders of magnitude. 10,11 Taking advantage of this property, we found that it proved possible to functionally characterize conformational states that are thermodynamically unstable or short-lived in solution. In particular, ligand binding to pure T and R states of Hb has been thoroughly investigated by resonance Raman spectroscopy, 12-17 CD, 18 and steady-state and time-resolved UV-vis absorption spectroscopy. 10,15-17,19-28 The emerging picture is that upon CO binding to T state Hb gels a slow, multiphasic process occurs, eventually leading to the appearance of spectroscopic markers usually attributed to the R state. 16 In their pioneering work, Shibayama and Saigo demonstrated that the R-to-T transition of oxy-Hb gels following reduction occurred with enormously slowed kinetics. 19 The slowing factor of the R-to-T transition rate was estimated to be on the order of 10 8 . The same slowing factor was found in a recent investigation of the events following CO binding to R and T state Hb gels, either in the absence (T - gels) or presence of allosteric effectors (T + gels). 24 It was shown that T + gels are characterized by kinetic binding properties of a pure T quater- nary state as determined in solution, R state gels by kinetic binding properties of a pure R quaternary state, and, remarkably, T - gels by a linear combination of T + and R kinetics. These ligand binding properties were interpreted using the tertiary two states (TTS) model recently proposed by Eaton and co-workers to explain the cooperative oxygen binding and heterotropic allosteric effects in Hb. 29,30 The TTS model overcomes the known deficiencies of the original Monod, Wyman, and Changeux (MWC) model, 31 and the modifications that followed, such as the Cooperon model of Brunori, 32,33 the model of Szabo and Karplus 34 based on the stereochemical mechanism of Perutz, and the generalization of the Szabo and Karplus model by Lee and Karplus. 35,36 The TTS model assumes that the quaternary T and R states of Hb are populated by two tertiary states, r and t, characterized by high and low ligand reactivity, respectively, which are functionally independent of the quaternary state of the molecule. The population of r and t states is regulated by quaternary structure and ligation and by allosteric heterotropic effectors. A key assumption is that the quaternary structure does not affect the reactivity of t and r, but rather affects their relative population. To test this hypothesis, we report herein a characterization of the T-to-R reaction pathway for T + and T - gels, demonstrat- ing that the observed complex kinetic time courses can be indeed * Corresponding author. Phone: +390521905208. Fax: +390521905223. E-mail: cristiano.viappiani@fis.unipr.it. † Dipartimento di Biochimica e Biologia Molecolare, Universita ` degli Studi di Parma, CNISM. ‡ Dipartimento di Fisica, Universita ` degli Studi di Parma, CNISM, and NEST CNR-INFM. J. Phys. Chem. B 2008, 112, 12790–12794 12790 10.1021/jp803040j CCC: $40.75  2008 American Chemical Society Published on Web 09/11/2008