Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

We have constructed a plasmid (pHE2) in which the synthetic human a-and 3-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed a and (3 chains of component a, respectively, have the expected masses.The remaining 10% of the P chain in component a corresponds in mass to the .8 chain plus methionine. In component b, both a and (3 chains have the correct masses without detectable N-terminal methionine (<2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the a and ,B chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the (3 chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.To make use of our ability to rationally design mutant human Hbs needed for research on structure-function relationships, an efficient expression system for producing unmodified human Hbs in high yields is needed. Human adult Hb (Hb A) is a tetrameric protein containing two a chains and two (3 chains having 141 and 146 amino acid residues each, respectively. Human globins and Hbs have been expressed in transgenic mice (1-4), transgenic swine (5), insect cell cultures (6), yeast (7, 8), and Escherichia coli (9-11). In many respects, the E. coli system is the best choice for our purposes because of its high expression efficiency and the ease ofperforming site-directed mutagenesis. The first E. coli system to express human a-and 3-globin as a fusion protein was developed by Nagai and Th0gersen (9,12), but the product processing procedure is very laborious and gives low yield. Thus, this expression system has limitations, especially when large amounts of recombinant Hb (rHb) are required for biochemical-biophysical studies. Hoffman et al.

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of 15 N-1 H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.A basic assumption in correlating protein structure and function is that the structure of a protein in the crystalline state is the same as that under physiological solution conditions. Although the weak intermolecular forces in a highly hydrated crystal are unlikely to shift ordered elements of a protein relative to one another, this does not necessarily hold true for flexible proteins that can switch between different conformations under allosteric control. For example, recent NMR data indicate that allosteric activation of the signaling protein NtrC simply involves the shift of a preexisting conformational equilibrium (1). The activated R-state conformation of the allosteric enzyme aspartate transcarbamoylase is another example where multiple conformations are sampled. The unliganded trimeric catalytic domain of this enzyme is fully active, but, except for increased intra-domain flexibility in the crystalline state, the trimer resembles the inactive T state of the holoenzyme (2). In addition, Alber, Schachman, and coworkers (3) found that upon ligation, this trimeric domain resembles the ligated holoenzyme, previously thought to represent the R state. This finding strongly suggests that the activated R state must be capable of traversing multiple conformations. The presence of such dynamic processes can easily be frozen out in the crystalline state.Human normal adult hemoglobin (Hb A) is the classic textbook example of a multimeric, allosteric protein and of the exquisite control a protein can exert over ligand binding. Hb A consists of four subunits: two ␣-chains of 141 amino acid residues each and two ␤-chains of 146 residues each. The oxygenation of Hb A in solution or inside red blood cells is cooperative; i.e., the binding of the first oxygen ligand to a Hb subunit enhances the binding of subsequent oxygen molecules to the remaining subunits. Oxygenation of ...