How nature tunes isoenzyme activity in the multifunctional catalytic globin dehaloperoxidase from Amphitrite ornata

The coelomic hemoglobin of Amphitrite ornata, termed dehaloperoxidase (DHP), is the first known multifunctional catalytic globin to possess biologically-relevant peroxidase and peroxygenase activities. Although the two isoenzymes of DHP, A and B, differ in sequence by only 5 amino acids out of 137 residues, DHP B consistently exhibits a greater activity than isoenzyme A. To delineate the contributions of each amino acid substitution to the activity of either isoenzyme, the substitutions of the five amino acids were systematically investigated, individually and in combination, using 22 mutants. Biochemical assays and mechanistic studies demonstrated that the mutants that only contained the I9L substitution showed increased i) k_cat values (peroxidase activity), ii) 5-Br-indole conversion and binding affinity (peroxygenase activity), and iii) rate of Compound ES formation (enzyme activation). Whereas the X-ray structures of the oxyferrous forms of DHP B (L9I) (1.96 Å), DHP A (I9L) (1.20 Å), and WT DHP B (1.81 Å) showed no significant differences, UV–visible spectroscopy (A_Soret/A₃₈₀ ratio) revealed that the I9L substitution increased the 5-coordinate high-spin heme population characterized by the “open” conformation (i.e., distal histidine swung out of the pocket), which likely favors substrate binding. The positioning of the distal histidine closer to the heme cofactor in the solution state also appears to facilitate activation of DHP via the Compound ES intermediate. Taken together, the studies undertaken here shed light on the structure-function relationship in dehaloperoxidase, but also help to establish the foundation for understanding how enzymatic activity can be tuned in isoenzymes of a multifunctional catalytic globin.     

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ～10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ～0.3 and ～0.8 ?, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.     

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.     

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.     

Assignment of heme and distal amino acid resonances in the 1H-NMR spectra of the carbon monoxide and oxygen complexes of sperm whale myoglobin

Assignments of resonances of the heme and distal amino acid protons in spectra of the CO and O2 complexes of sperm whale myoglobin are reported. These resonances provide information on the conformation of the heme pocket. For oxymyoglobin, the assignments of the heme meso protons disagree with those proposed previously on the basis of partial deuteration experiments. Rapid ring flips about the C beta-C gamma bond are detected for Phe-CD1. Recent claims for two conformational substates of valine-E11 in carbonmonoxymyoglobin (Bradbury, J.H. and Carver, J.A. (1984) Biochemistry 23, 4905-4913) are shown to be in error. The pK of His-97 (FG3) in carbonmonoxymyoglobin has been determined (pK = 5.9). This residue appears to influence many spectroscopic properties of myoglobin. The distal His-E7 in carbonmonoxymyoglobin has pK less than 5.0. Differences in the heme pocket conformation in the CO complexes of myoglobin and leghemoglobin are discussed. These differences may be influential in O2 and CO association reactions.     

Structural and dynamic repercussions of heme binding and heme-protein cross-linking in Synechococcus sp. PCC 7002 hemoglobin

The recombinant two-on-two hemoglobin from the cyanobacterium Synechoccocus sp. PCC 7002 (S7002 rHb) is a bishistidine hexacoordinate globin capable of forming a covalent cross-link between a heme vinyl and a histidine in the C-terminal helix (H helix). Of the two heme axial histidines, His46 (in the E helix, distal side) and His70 (in the F helix, proximal histidine), His46 is displaced by exogenous ligands. S7002 rHb can be readily prepared as an apoglobin (apo-rHb), a non-cross-linked hemichrome (ferric iron and histidine axial ligands, rHb-R), and a cross-linked hemichrome (rHb-A). To determine the effects of heme binding and subsequent cross-linking, apo-rHb, rHb-R, and rHb-A were subjected to thermal denaturation and 1H/2H exchange. Interpretation of the latter data was based on nuclear magnetic resonance assignments obtained with uniformly 15N- and 13C,15N-labeled proteins. Apo-rHb was found to contain a cooperative structural core, which was extended and stabilized by heme binding. Cross-linking resulted in further stabilization attributed mainly to an unfolded-state effect. Protection factors were higher at the cross-link site and near His70 in rHb-A than in rHb-R. In contrast, other regions became less resistant to exchange in rHb-A. These included portions of the B and E helices, which undergo large conformational changes upon exogenous ligand binding. Thus, the cross-link readjusted the dynamic properties of the heme pocket. 1H/2H exchange data also revealed that the B, G, and H helices formed a robust core regardless of the presence of the heme or cross-link. This motif likely encompasses the early folding nucleus of two-on-two globins.     

Internal Binding of Halogenated Phenols in Dehaloperoxidase-Hemoglobin Inhibits Peroxidase Function

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

Proline 107 is a major determinant in maintaining the structure of the distal pocket and reactivity of the high-spin heme of MauG

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H(2)O(2) and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH

Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7, 8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676), Trp(678), Phe(691), His(692), and Tyr(706), together with the neighboring Arg(414) residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH. Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/l-Arg binding and/or electron transfer to the heme from NADPH. The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(414) to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH. Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/l-Arg binding and/or efficient electron transfer to occur.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner

The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).     

Crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate. Conformational change of the distal helix during the heme cleavage reaction

The crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate (biliverdin(Fe)-HO-1), the immediate precursor of the final product, biliverdin, has been determined at a 2.4-A resolution. The electron density in the heme pocket clearly showed that the tetrapyrrole ring of heme is cleaved at the alpha-meso edge. Like the heme bound to HO-1, biliverdin-iron chelate is located between the distal and proximal helices, but its accommodation state seems to be less stable in light of the disordering of the solvent-exposed propionate and vinyl groups. The middle of the distal helix is shifted away from the center of the active site in biliverdin(Fe)-HO-1, increasing the size of the heme pocket. The hydrogen-bonding interaction between Glu-29 and Gln-38, considered to restrain the orientation of the proximal helix in the heme-HO-1 complex, was lost in biliverdin(Fe)-HO-1, leading to relaxation of the helix. Biliverdin has a distorted helical conformation; the lactam oxygen atom of its pyrrole ring-A interacted with Asp-140 through a hydrogen-bonding solvent network. Because of the absence of a distal water ligand, the iron atom is five-coordinated with His-25 and four pyrrole nitrogen atoms. The coordination geometry deviates considerably from a square pyramid, suggesting that the iron may be readily dissociated. We speculate that the opened conformation of the heme pocket facilitates sequential product release, first iron then biliverdin, and that because of biliverdin's increased flexibility, iron release triggers its slow dissociation.     

Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1

We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.     

Comparison of the heme-free and -bound crystal structures of human heme oxygenase-1

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin. The crystal structure of human HO-1 in complex with heme reveals a novel helical structure with conserved glycines in the distal helix, providing flexibility to accommodate substrate binding and product release (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). To structurally understand the HO catalytic pathway in more detail, we have determined the crystal structure of human apo-HO-1 at 2.1 A and a higher resolution structure of human HO-1 in complex with heme at 1.5 A. Although the 1.5-A heme.HO-1 model confirms our initial analysis based on the 2.08-A model, the higher resolution structure has revealed important new details such as a solvent H-bonded network in the active site that may be important for catalysis. Because of the absence of the heme, the distal and proximal helices that bracket the heme plane in the holo structure move farther apart in the apo structure, thus increasing the size of the active-site pocket. Nevertheless, the relative positioning and conformation of critical catalytic residues remain unchanged in the apo structure compared with the holo structure, but an important solvent H-bonded network is missing in the apoenzyme. It thus appears that the binding of heme and a tightening of the structure around the heme stabilize the solvent H-bonded network required for proper catalysis.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.