Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V-max) of 670 s(-1) and K-m values for NADH, NO, and O-2 equal to 4.8, 0.28, and similar to 100 mu M, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V-max is similar to 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of similar to 15 mu M-1 s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 mu M-1 s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O-2, dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O-2] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O-2 dissociation rate constant similar to 80-fold, and reduces NO dioxygenase activity similar to 30-fold, demonstrating the importance of the tyrosine hydroxyl for O-2 affinity and NO scavenging activity. At 37 degrees C, V-max/K-m(NO) is 2,400 mu M-1 s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group.Globin side chains at the Leu 89 (F4), His 97 (FG3), Ile 99 (FG5), and Leu 104 (G5) positions on the proximal side of the heme pocket strongly influence heme affinity.The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography.Leu 89 and His 97 are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring.Ile 99 and Leu 104 are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group.The apolar amino acids Leu 89 , Ile 99 , and Leu 104 "waterproof " the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment.Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss.Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His 93 -Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

Cyanide binding to myoglobin is much slower than that of other ferric and ferrous ligands, suggesting rate limitation by bond formation and disruption within the distal pocket. This interpretation is supported by two key experimental observations. First, His64(E7) to Gly and Ala mutations, which open a direct channel from the solvent to the iron atom, and Phe46(CD4) to Leu, Ile, and Val mutations, which increase the mobility of the distal histidine, have little effect on the association rate constant for cyanide binding. In contrast, these mutations cause 100-1000-fold increases in the rate constant for azide binding, showing convincingly that the binding of this ligand is limited by the rate of its movement into the protein. Second, the rate constant for cyanide dissociation is unaffected by changing the size of the residue at position 64(E7) in the series Gly, Val, Leu, Ile, Phe, whereas there is a 2000-fold decrease in the rate of azide dissociation in going from Gly64 to Phe64 metmyoglobin. The major determinants of the cyanide affinity are the ease of water displacement from the ferric iron atom in metmyoglobin, the acid dissociation constant of HCN inside the protein (K*a), and steric hindrance and electrostatic interactions at the sixth coordination position. Direct hydrogen bonding to the distal histidine does not appear to play an important role in stabilizing bound cyanide. Instead, the general polarity of the distal pocket and its effect on K*a are the key factors regulating cyanide affinity under physiological conditions.

The structures of the deoxy, oxy, and aquomet forms of native sperm whale myoglobin reconstituted with cobalt protoporphyrin IX have been determined by x-ray crystallography. As expected, cobalt myoglobin closely resembles native iron myoglobin in overall structure, especially in their respective aquomet forms. In the cobalt oxymyoglobin structure, the Nε of distal histidine 64 lies within hydrogen bonding distance to both the oxygen atom directly bonded to the cobalt and the terminal oxygen atom, in agreement with previous EPR and resonance Raman studies. The metal atom in cobaltous myoglobin does show a small 0.06-Å out-of-porphyrin plane displacement when moving from the oxy to deoxy state. In the case of the native iron-containing myoglobin, the oxy to deoxy transition results in a larger 0.16-Å displacement of the metal farther out of the porphyrin plane, attributed to an increase in spin from S = 0 to S = 2. The small displacement in cobalt myoglobin is due to a change in coordination geometry, not spin state (S = 1/2 for both cobalt deoxy- and oxymyoglobin). The small out-of-porphyrin plane movement of cobalt which accompanies deoxygenation of myoglobin also occurs in cobalt hemoglobin and serves to explain why cooperativity, although reduced, is still preserved when iron is replaced by cobalt in human hemoglobin.

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.

The structural factors governing azide and cyanide binding have been examined by measuring the effects of 46 mutations at key topological positions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His64 (E7) with smaller amino acids results in dramatic increases in the association rate constant for azide binding primarily due to relief of steric hindrance imposed by the imidazole side chain. Gln64 and His64 (native) metmyoglobins have abnormally low rate constants for azide dissociation (0.1-0.3 s-1) due to direct hydrogen bonding between the N epsilon atoms of these residues and the bound ligand. Mutations at positions 67(E10) and 68(E11) produce large but complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, influence the rate of anion movement into the distal pocket, and affect the stability of the Fe-N3 bond. Replacement of Phe46 with Leu or Val and substitution of Arg(Lys)45 with Glu and Ser cause disorder in the position of the distal histidine side chain and result in 4-700-fold increases in both k'N3 and kN3 but produce little change in overall azide affinity. All of these results suggest strongly that azide enters the distal pocket of native myoglobin through a polar channel that is regulated by a His64 "gate." In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the distal histidine is replaced with apolar residues. His64, Gln64, and distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.

There are at least two picosecond kinetic components in the rebinding of NO to native sperm whale myoglobin. Petrich et al. (Petrich, J. W., Lambry, J.-C., Kuczera, K., Karplus, M., Poyart, C., and Martin, J.-L. (1991) Biochemistry 30, 3975-3987) attribute the slowing of the reaction to a movement of the iron atom out of the plane of the heme following ligand dissociation. In contrast, Gibson et al. (Gibson, Q. H., Regan, R., Elber, R., Olson, J. S., and Carver, T. E. (1992) J. Biol. Chem. 267, 22022-22034) have explained multiphasic geminate reactions by diffusion of NO into the distal heme pocket as determined by its detailed structure. O2 and NO rebinding to iron and cobalt derivatives of native, V68F, and V68I sperm whale myoglobin has been examined. Each iron protein shows a biphasic time course of NO rebinding reactions with widely different rates and amplitudes. Although cobalt does not move out of the plane of the porphyrin on ligand removal, the reactions of the iron and cobalt derivatives of each protein were closely similar. The time course of O2 rebinding to cobalt was also similar to that of NO rebinding to iron. These results are consistent with a primary role for the structure of the distal pocket in determining diffusion of ligands away from the metal atom and as a result the time course of picosecond ligand rebinding.