Published: February 18, 2011 r2011 American Chemical Society 2436 dx.doi.org/10.1021/jp110833v | J. Phys. Chem. B 2011, 115, 2436–2446 ARTICLE pubs.acs.org/JPCB Kinetics of Carbon Monoxide Migration and Binding in Solvated Neuroglobin As Revealed by Molecular Dynamics Simulations and Quantum Mechanical Calculations Massimiliano Anselmi,* ,† Alfredo Di Nola, † and Andrea Amadei ‡ † Department of Chemistry, University of Rome “La Sapienza”, Rome, Italy ‡ Department of Chemistry, University of Rome “Tor Vergata”, Rome, Italy b S Supporting Information ABSTRACT: Neuroglobin (Ngb) is a globular protein that reversibly binds small ligands at the six coordination position of the heme. With respect to other globins similar to myoglobin, Ngb displays some peculiarities as the topologi- cal reorganization of the internal cavities coupled to the sliding of the heme, or the binding of the endogenous distal histidine to the heme in the absence of an exogenous ligand. In this Article, by using multiple (independent) molecular dynamics trajectories (about 500 ns in total), the migration pathways of photolized carbon monoxide (CO) within solvated Ngb were analyzed, and a quantitative description of CO migration and corresponding kinetics was obtained. MD results, combined with quantum mechanical calculations on the CO-heme binding-unbinding reaction step in Ngb, allowed construction of a quantitative model representing the relevant steps of CO migration and rebinding. ’ INTRODUCTION The ligand diffusion in the interior of globins has been extensively investigated in recent years with several experimental and computational techniques. The major contribution for the knowledge of such a topic has come from the study of myoglobin (Mb). In fact, the small size, the relative structural stability, and the complex functional behavior make myoglobin a perfect model system to investigate, at the atomic level, protein bio- chemical activities. Time-resolved X-ray diffraction, 1-5 Fourier transform infrared spectroscopy, 6-9 kinetic measurements, 10,11 and theoretical approaches 12-18 are usefully adopted to study the diffusion of small diatomic ligands, such as O 2 , CO, and NO, in wild-type Mbs and their mutants. However, a few years ago, neuroglobin (Ngb), a new member of the globin family, was discovered to be markedly expressed in the brain. 19 Like Mb, Ngb is a monomeric protein, which reversibly binds small ligands at the sixth coordination position of the heme, and, despite its small sequence similarity with other globins, it displays the typical globin fold; consequently, Ngb conserves some canonical residues, among others a proximal histidine that steadily binds to the heme iron and a distal histidine facing the binding site. 20,21 In the ferrous form, Ngb binds O 2 , CO, and NO, but, in contrast to Mb, in the absence of an exogenous ligand, both the ferric and the ferrous forms are hexacoordinated to the proximal and distal histidines. 22,23 Such a behavior implies a competition between an exogenous ligand and the distal histidine for the binding at the sixth coordination position on the heme. The physiological role of Ngb is still unknown, even though convincing experimental evidence shows that it is involved in the regulation of a still unclear neuronal protective mechanism under hypoxia and ischemia. 24,25 Experimental observations show that Ngb interacts with the R-subunit of heterotrimeric G Rβγ protein, enforcing the hypothesis, which assigns to Ngb a role of signal transducer. 26-28 Recently, it has been suggested that Ngb inhibits the intrinsic pathway of apoptosis in vitro and may prevent the activation of pro-caspase 9 by interaction of cyto- chrome c. 29 Moreover, it is worth reminding that the average Ngb concentration seems too low (<1 μM) to play a Mb-like role in the transport and storage of O 2 , although this function is not yet excluded. Therefore, Ngb and Mb similarity makes such a protein an alternative and complementary system in the study of the globins, whereas its peculiarities represent a new challenge for the interpretation of the relation between structure and function. The three-dimensional structure was solved by X-ray diffraction for the unliganded Ngb from human and mouse 20,21 and for ferrous CO-bound Ngb. 30 Comparison of the crystal structures of two murine Ngb redox states 21,30 showed that binding of CO is associated to structural changes involving a significant heme sliding and a topological reorganization of the internal cavities. 30 Such a heme sliding was also detected with Molecular Dynamics Received: November 12, 2010 Revised: January 17, 2011