The flavoHbs 1 belong to a 1.8 billion-year-old family of globin molecules that includes O 2 -binding Hbs and Mbs isolated from animals, plants, fungi, protozoa, bacteria, and worms (1-6). FlavoHbs have a unique two-domain structure containing linked Hb and reductase domains with extensive homology to the mammalian Hbs and metHb reductases (1, 7). Other Hbs appear to be co-expressed with associated metHb reductases (8). An O 2 transport or storage function, like that of the erythrocytic Hbs and muscle Mbs, has been suggested for some microbial and plant (flavo)Hbs (4, 9); however, other functions including the catalysis of oxidations have long seemed more likely (10 -12).Recently, we described an NO dioxygenase (NOD) produced by Escherichia coli that utilizes O 2 and NAD(P)H to convert NO to nitrate (Equation 1) and identified it as a flavoHb (13,14). Subsequent studies have shown that related bacterial and yeast flavoHbs display a similar NOD activity.A role for flavoHbs in NO detoxification is supported by the ability of flavoHbs to protect bacteria against NO or nitroso compounds (13, 14, 16 -18) and by their induction in bacteria exposed to NO, nitrate, nitrite, or nitroso compounds (13, 14, 17, 19 -22). However, the mechanism of NO detoxification, and thus the function of the flavoHbs, is obscured by the possibility of multiple reaction mechanisms involving NO. Other NO detoxifying activities for flavoHbs, including denitrosylation of nitrosothiols (17), NO reduction (17, 23), and NO sequestration (16, 23), have been offered to explain the protection flavoHbs afford to bacteria against NO and nitrosoglutathione. Thus, an understanding of the biological function(s) of the flavoHbs demands a greater knowledge of their various activities.In this report, steady-state, reduction, and ligand binding kinetics of the E. coli NOD (flavoHb) were measured in order to define its function and the mechanism of NO dioxygenation. We also examined the effects of amino acid substitutions at the highly conserved Tyr(B10) position on NOD activity, reduction, and ligand binding kinetics (7,24). Key differences between flavoHb and other Hbs are discussed in light of this specialized but perhaps ancient NO dioxygenation and detoxification function of hemoglobin. MATERIALS AND METHODSCells and Reagents-The flavoHb-deficient E. coli strain RB9060 (25) was generously provided by Alex Ninfa (University of Michigan). Plasmid pAlter containing the E. coli hmp gene was prepared as described previously (13). FAD, NADPH, and bovine hemin were purchased from Sigma. NADH, bovine liver catalase (260,000 units/ml), and deoxyribonuclease were obtained from Roche Molecular Biochemicals. Saturated NO was prepared as described previously (26). Saturated O 2 (1.14 mM) was prepared by vigorously scrubbing a solution of 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA (buffer A) at 25°C and atmospheric pressure with 99.993% O 2 (Praxair, Bethlehem, PA) in a rubber septum-sealed glass vial vented with a syringe needle. Manganese-containing...

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

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UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4°C. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known fivecoordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (Fe III PPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of Fe III PPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.Heme proteins with protein-derived oxygen donor proximal ligands are relatively rare in nature. The best known example of such ligation is that of bovine liver catalase which contains a tyrosine phenolate proximal heme iron ligand (1).In addition, a series of naturally occurring hemoglobin mutants having distal or proximal histidines replaced by tyrosine or glutamate (the M hemoglobins) have been established by X-ray crystallography (2-4) and resonance Raman spectroscopy (5, 6) to have phenolate or carboxylate ligation. Other proteins with tyrosinate and glutamate oxygen donor ligation have been recently produced by site-directed mutagenesis of various myoglobins (7-10) and cytochrome c peroxidase (11).The use of site-directed mutagenesis techniques has become invaluable in identifying catalytically and structurally important protein residues within a protein system. A relatively new type of mutation, based on altering the size of the amino acid at the point of mutation, substitutes a glycine or alanine for the larger original residue and leaves a cavity within the protein. Termed cavity mutants, these mutated proteins demonstrate the ability to employ exogenous ligands to reconstitute their wild-type activity. This rescue of activity has been seen for the cavity mutants of azurin (12), carbonic anhydrase (13), hexose-1-phosphate uridyltransferase (14), and various heme proteins (15-18).