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F S Mathews

Publications and source records attributed to F S Mathews.

At least 37 records · Page 2Linked to original sources
PubMed1998

Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A resolution reveals the active conformation.

BACKGROUND: Copper-containing amine oxidases (CAOs) are widespread in nature. These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. The enzyme is dimeric with a subunit molecular weight of 78 kDa. Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency. RESULTS: The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging. The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme. Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent. CONCLUSIONS: The structure shows TPQ in a position poised for catalysis. This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Crystallographic study of azurin from Pseudomonas putida.

Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier. Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit. The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 A, beta = 107.79 degrees, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 A, beta = 103.12 degrees. The latter crystals were found to have four zinc ions incorporated into the crystal lattice. Both crystal structures were solved by the molecular-replacement method using the program MERLOT. The search model was the structure of azurin from Alcaligenes denitrificans. The crystallographic R factor for native azurin is 0.169 (Rfree = 0. 257) from 8 to 1.92 A resolution, while that for zinc azurin is 0. 181 (Rfree = 0.248) from 10 to 1.6 A resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 A. In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with Ndelta1 from His46 and His117, and one with the thiolate S atom of Cys112. Two longer axial approaches are made by the Sgamma from Met121 and the carbonyl O atom from Gly45. This results in a distorted trigonal bipyramidal co-ordination around the Cu atom. It further confirms the presence of a weak fifth bond to the copper in P. putida azurin, as with other azurin structures described at high resolution. The Ndelta1 atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans. In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain. In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules. The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site. The zinc ligands are Ndelta from His, carboxylate O atoms from Asp and Glu, Ogamma from Ser and water molecules. One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (-101) direction and is primarily responsible for the altered unit-cell parameters. Two of the other zinc ions bind to His83, one in each molecule.

Amino Acid Sequence↗
PubMed1998

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans has been refined at 1.75 A resolution utilizing the DNA-based protein sequence. The final model incorporates 8034 atoms per molecule, including 552 molecules of solvent, and gives an R-factor of 0.163. The molecule is an H2L2 hetero-tetramer containing a non-crystallographic 2-fold axis of symmetry. The 373-residue H subunit is folded into seven repeats of a four-stranded antiparallel beta-sheet motif, arranged in a propeller-like pattern about a pseudo-7-fold rotational axis of symmetry. Each L subunit contains 131 residues folded in a tight structure composed of five beta-strands in two sheets and crosslinked by six disulfide bonds. In addition there is an intrasubunit covalent linkage between two tryptophan side-chains that form the unique redox center, tryptophan tryptophylquinone (TTQ). The active site contains the O-6 carbonyl of TTQ, the side-chains of Asp32L Asp76L, Tyr119L and Thr122L, and two solvent molecules. A potential "gate" (Phe55H) separates the closed active-site cavity from a channel containing a group of highly ordered water molecules to bulk solvent. Phe55H and Tyr119L, and a number of neighboring oxygen atoms, may also provide a binding site for monovalent cations that are known to affect the reactivity and spectral properties of TTQ as well as the oxidative half reaction. The overall reaction has been dissected into a number of discrete steps that may require participation by several individual amino acid residues in the active site acting as general acids and bases.

Amino Acid Sequence↗
PubMed1998

HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense.

HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues. Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands. Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast. Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.

Amino Acid Sequence↗
PubMed1997

Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis.

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99. Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.

Amino Acid Sequence↗
PubMed1997

Crystallographic study of yeast copper amine oxidase.

The copper-containing amine oxidase from the yeast Hansenula polymorpha (YAO) has been crystallized and partially solved by molecular replacement. It catalyzes the oxidative deamination of primary amines by molecular oxygen to the corresponding aldehydes, ammonia and hydrogen peroxide. It contains a covalently bound redox cofactor, topa quinone, generated by post-translational modification of a single tyrosine side chain. The crystals of YAO are orthorhombic, with space-group symmetry P2(1)2(1)2(1) and unit-cell dimensions a = 138.8, b = 148.2, c = 234.0 A and diffract X-rays beyond 2.0 A resolution. Solution by molecular replacement using the E. coli amine oxidase structure [Parsons, Convery, Wilmot, Yadav, Blakeley, Corner, Philips, McPherson & Knowles (1995). Structure, 3, 1171-1184] as a search model reveals that there are three dimers in the asymmetric unit in a trigonal arrangement having 32 point-group symmetry. The solution agrees well with the self-rotation function of YAO. The non-crystallographic threefold axis lies parallel to a crystallographic twofold screw axis and each dimer has twofold symmetry. Phases from the refined model based on the molecular-replacement solution were used to solve one heavy-atom derivative. Model building from the unbiased isomorphous replacement phases is in progress.

Journal Article↗
PubMed1997

Selective modification of alkylammonium ion specificity in trimethylamine dehydrogenase by the rational engineering of cation-pi bonding.

In trimethylamine dehydrogenase (TMADH), substrate is bound in the active site by organic cation-pi bonding mediated by residues Tyr-60, Trp-264, and Trp-355. In the closely related dimethylamine dehydrogenase (DMADH), modeling suggests that a mixture of cation-pi bonding and conventional hydrogen bonding is responsible for binding dimethylamine. The active sites of both enzymes are highly conserved, but three changes in amino acid identity (residues Tyr-60 --> Gln, Ser-74 --> Thr, and Trp-105 --> Phe, TMADH numbering) were identified as probable determinants for tertiary --> secondary alkylammonium ion specificity. In an attempt to switch the substrate specificity of TMADH so that the enzyme operates more efficiently with dimethylamine, three mutant proteins of TMADH were isolated. The mutant forms contained either a single mutation (Y60Q), double mutation (Y60Q x S74T) or triple mutation (Y60Q x S74T x W105F). A kinetic analysis in the steady state with trimethylamine and dimethylamine as substrate indicated that the specificity of the triple mutant was switched approximately 90,000-fold in favor of dimethylamine. The major component of this switch in specificity is a selective impairment of the catalytic efficiency of the enzyme with trimethylamine. Rapid-scanning and single wavelength stopped-flow spectroscopic studies revealed that the major effects of the mutations are on the rate of flavin reduction and the dissociation constant for substrate when trimethylamine is used as substrate. With dimethylamine as substrate, the rate constants for flavin reduction and the dissociation constants for substrate are not substantially affected in the mutant enzymes compared with wild-type TMADH. The results indicate a selective modification of the substrate-binding site in TMADH (that impairs catalysis with trimethylamine but not with dimethylamine) is responsible for the switch in substrate specificity displayed by the mutant enzymes.

Alkylation↗
PubMed1997

An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: kinetics of transfer in wild-type and mutant complexes.

In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.

Bacteria↗
PubMed1996

Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine.

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

Apoenzymes↗
PubMed1996

X-ray structure of the cupredoxin amicyanin, from Paracoccus denitrificans, refined at 1.31 A resolution.

High-resolution X-ray diffraction data to d(min) = 1.31 A were collected on a Xuong-Hamlin area detector from crystals of the blue-copper protein amicyanin, isolated from P. denitrificans. With coordinates from the earlier 2.0 A structure determination as a starting point, simulated annealing and restrained positional and temperature factor refinements using the program X-PLOR resulted in a final R factor of 15.5%, based on 21 131 unique reflections in the range 8.0-1.3 A. Comparison of the 1.31 A structure with that at 2.0 A shows the same basic features. However, the high-resolution electron-density maps clearly reveal additional solvent molecules and significant discrete disorder in protein side chains and within the solvent structure. As a consequence of modelling side-chain disorder, several new hydrogen-bonding interactions were identified.

Journal Article↗
PubMed1996

Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1.

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophus W3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca2+ and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cis peptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold beta-propeller, each "blade" of which is a four-stranded antiparallel twisted beta-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four beta-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold beta-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight beta-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the beta-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent beta-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca2+ is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.

Alcohol Oxidoreductases↗
PubMed1996

Enzymatic and electron transfer activities in crystalline protein complexes.

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme.

Bacterial Proteins↗
PubMed1996

Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution.

The structure of bovine liver cytochrome b(5), a soluble 93-residue proteolytic fragment of a 16 kDa membrane-bound hemoprotein, initially solved at 2.0 A resolution, has been refined at 1.5 A using data collected on a diffractometer. Refinement to 2.0 A resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 A resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5 A. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R = 0.164 for the data between 1.5 and 10 A.

Journal Article↗
PubMed1996

Covalent structure of the flavoprotein subunit of the flavocytochrome c: sulfide dehydrogenase from the purple phototrophic bacterium Chromatium vinosum.

The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c-sulfide dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three-dimensional structure determination (Chen Z et al., 1994, Science 266:430-432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 +/- 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three-dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three-dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Trp 391, Glu 167, and the positive end of a helix dipole.

Amino Acid Sequence↗
PubMed1996

Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs.

The X-ray structure of the homotetrameric lysosomal acid hydrolase, human beta-glucuronidase (332,000 Mr), has been determined at 2.6 A resolution. The tetramer has approximate dihedral symmetry and each promoter consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179-204 form a beta-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.

Amino Acid Sequence↗
PubMed1995

The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase.

When p-cresol methylhydroxylase (PCMH) is expressed in its natural host Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escherichia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr384 of the alpha subunit and the correct alpha 2 beta 2 form of the enzyme is assembled. The apoflavoprotein has been expressed in E. coli in the absence of the beta cytochrome c subunit and purified. While noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs. Covalent flavinylation requires one molecule each of FAD and cytochrome for each flavoprotein subunit. The flavinylation process leads to the 2-electron-reduced form of covalently bound FAD, and the resulting alpha 2 beta 2 enzyme is identical to wild-type PCMH. This work presents clear evidence that covalent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation of FAD or of the protein. This work is the first to define the basic chemistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mechanism of this process. It also demonstrates, for the first time, the absolute requirement for a partner subunit in the post-translational modification of a protein. It is proposed that the covalent FAD bond to Tyr384 and the phenolic portion of this Tyr are part of the essential electron transfer path from FAD to heme.

Amino Acid Sequence↗
PubMed1995

Subunit interactions in Ascaris hemoglobin octamer formation.

The oxygen-avid, perienteric hemoglobin of Ascaris is a homooctamer. Each subunit contains two tandem globin domains that are highly homologous with the exception of a charged COOH-terminal extension. In solution, recombinant domain one (D1) exists as a monomer, whereas recombinant domain two with the COOH-terminal tail (D2) is primarily an octamer. To examine the role of the COOH-terminal extension in Ascaris hemoglobin multimer formation, we attached the tail to the monomeric, heme-containing proteins, myoglobin and D1; neither construct was capable of multimer formation. Additionally, we removed the tail from both full-length Ascaris hemoglobin and D2. This substantially decreased, but did not eliminate, multimerization. We further characterized subunit interactions by disrupting full-length hemoglobin multimers with the chaotropic salt, NaSCN, which yielded intermediate oligomers. In solution, D2 demonstrated a greater propensity to dissociate than full-length hemoglobin, indicating that D1 contributes to octamer stability. D1 formed a weak dimer in its crystal; thus, we analyzed interactions along the subunit interface. Hydrogen bonds as well as hydrophobic and electrostatic forces appeared to contribute to dimer formation. Amino acid substitutions along this interface in D2 are predicted to enhance subunit interactions for that domain. Our studies reveal that the COOH-terminal tail is necessary, but not sufficient, for efficient octamer formation. Other regions, possibly with the E- and F-helices and AB loops of both domains, appear to be important for Ascaris hemoglobin octamer formation.

Amino Acid Sequence↗