The X-ray Structure of Ferric Escherichia coliFlavohemoglobin Reveals an Unexpected Geometry of the Distal Heme Pocket

Abstract

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 Å and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.Flavohemoglobins are oxygen-binding proteins composed of a typical globin domain containing a B-type heme fused with a ferredoxin reductase-like FAD-and NAD-binding domain. Together with single domain bacterial hemoglobins and six-helices containing "truncated hemoglobins," flavohemoglobins are part of a vast family of "hemoglobin-like" proteins whose functions are still elusive. Because of the identification of flavohemoglobin genes in a wide variety of prokaryotic and eukaryotic microorganisms, a crescendo of experimental observations has revealed that these proteins possess unique structural and functional properties unrelated to those of hemoproteins involved in oxygen transport and storage (1).Escherichia coli flavohemoglobin (HMP) 1 is certainly the most extensively studied protein within the bacterial hemoglobin family. At present, the most credited physiological function of HMP has been inferred from the observation that protein expression in the bacterial cell is enhanced in the presence of nitric oxide releasers in the culture medium (2-4). Accordingly, HMP has been shown to be able to catalyze the oxidation of free nitric oxide to nitrate both in vivo and in vitro in the presence of oxygen and NADH (5). In this framework, HMP is thought to be involved in the response of the bacterial cell to the potentially harmful role of free nitric oxide in giving rise to nitrosative stress. Nevertheless, although the nitric-oxide dioxygenase activity stands as the major functional hypothesis for HMP, the molecular mechanism and the structural determinants at the basis of this enzymic function remain poorly understood. In particular, it has not been established as yet how the amino acid side chains in the distal heme pocket affect...

Thermoglobin, Oxygen-avid Hemoglobin in a Bacterial Hyperthermophile

“…Crystal structures of bacterial Hbs similar to AaTgb, V. stercoraria Hb (47,48), A. eutrophus Fhb (49, 50), E. coli Fhb (51), and the Hb domain of Bacillus subtilis HemAT (33), do not consistently reveal a distal binding site similar to that of A. suum Hb. In cases where direct interaction between the B10 tyrosine and bound ligand cannot be observed or deduced, the crystallized biochemical states do not contain the appropriate gas ligands that may order the B10 tyrosine and E7 glutamine side chains.…”

A TyrCD1/TrpG8 hydrogen bond network and a TyrB10—TyrCD1 covalent link shape the heme distal site of Mycobacterium tuberculosis hemoglobin O

“…Remarkably, residue CD1, which is invariably Phe in (non)vertebrate Hbs and Mbs, as well as in trHbs from groups I and III, is Tyr in trHbO and in other trHbs from group II. As observed for all currently known group II and III trHbs, trHbO displays a Trp residue at the G8 site, where smaller apolar residues (Val, Leu, or Ile) are constantly found in group I trHbs, as well as in (non)vertebrate Hbs͞Mbs, flavohemoglobins, and plant Hbs (1,4,10,(18)(19)(20). Resonance Raman data indicate that in trHbO, a hydrogen-bonded network stabilizes the hemebound O 2 and CO ligands, as evidenced by frequencies of the Fe-O 2 and Fe-CO stretching modes (13).…”

“…Crystal structures of three group I trHbs (2,3) revealed that trHbs are clearly not just another variation on the motif of vertebrate myoglobin (Mb) and Hb. Neither are they similar to nonvertebrate Hbs, including the heme-containing domain of flavohemoglobins, nor to the plant symbiotic and nonsymbiotic Hbs (4)(5)(6)(7)(8)(9)(10). Major structural differences associated with known trHbs are an unprecedented 2-on-2 ␣-helical sandwich fold, resulting from striking editing of the classical 3-on-3 globin ␣-helical sandwich, and an extended hydrophobic tunnel͞cavity network linking the solvent space and the distal heme pocket (1)(2)(3).…”

“…The different H-bond networks thus achievable may not only stabilize the bound O 2 but also modulate the positioning and dynamics of distal pocket residues that participate in the control of ligand access to the iron, ligand diffusion within the distal heme pocket, and ligand diffusion into and out of the distal heme pocket (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).…”

Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

“…It has been detected in some myoglobin distal mutants (4,5), the monomeric flavoHb (6), and the dimeric Scapharca inaequivalvis Hb (7). More recently, the 5c species has been found in Hbs with higher structural complexity, such as tetrameric Hbs from polar fish (8-10) and giant Hbs (11).…”