Themiste Zostericola Research Articles

Magnetic Circular Dichroism Study of a Dicobalt(II) Complex with Mixed 5- and 6-Coordination: A Spectroscopic Model for Dicobalt(II) Hydrolases

The magnetic circular dichroism (MCD) study of [Co(2)(mu-OH)(mu-Ph(4)DBA)(TMEDA)(2)(OTf)], in which Ph(4)DBA is the dinucleating bis(carboxylate) ligand dibenzofuran-4,6-bis(diphenylacetate) and TMEDA is N,N,N',N'-tetramethylethylenediamine, is presented. This complex serves as an excellent spectroscopic model for a number of dicobalt(II) enzymes and proteins that have both the mu-hydroxo, mu-carboxylato bridging and asymmetric 6- and 5-coordination. The low-temperature MCD spectrum of the model complex shows bands at 490, 504, and 934 nm arising from d-d transitions on the 6-coordinate Co(II) and bands at 471, 522, 572, 594, and 638 nm arising from d-d transitions on the 5-coordinate Co(II). The most intense MCD bands are at 504 and 572 nm for 6- and 5-coordinate Co(II), respectively, and these two bands are found in the MCD spectra of dicobalt(II)-substituted methionine aminopeptidase from Escherichia coli (CoCoMetAP), glycerophosphodiesterase from Enterobacter aerogenes (CoCoGpdQ), aminopeptidase from Aeromonas proteolytica (CoCoAAP), and myohemerythrin from Themiste zostericola (CoCoMyoHry). These dicobalt(II)-substituted proteins are known to have one 5- and one 6-coordinate Co(II) bridged by one or two carboxylates and either a water or a hydroxide. The uncertainty of the bridging water's state of protonation is problematic, as this is a likely candidate for the attacking nucleophile in the dimetallohydrolases. Analysis of the variable-temperature variable-field (VTVH) MCD data determined that the Co(II) ions in the model complex are ferromagnetically coupled with a J of 3.0 cm(-1). A comparison of all dicobalt(II) complexes and dicobalt(II)-substituted protein active sites with the mu-hydroxo/aqua, mu-carboxylato bridging motif reveals that J is either zero or negative (antiferromagnetic) in the mu-aqua systems and positive (ferromagnetic) in the mu-hydroxo systems. It was also determined that the Co(II) ions in CoCoAAP and CoCoMyoHry are ferromagnetically coupled, each with a J of 3.4 cm(-1), which suggests that these ions have a mu-hydroxo bridging ligand.

Molecular Evolution and Phylogeny of Sipunculan Hemerythrins

We sequenced seven new hemerythrin (Hr) and myohemerythrin (myoHr) cDNAs from Sipunculus nudus and Golfingia vulgaris vulgaris, thus providing new comparative data that significantly increase the set of the known Hr and myoHr sequences. Bayesian inference, maximum likelihood, and maximum parsimony phylogenetic analyses were performed to investigate the evolutionary relationships among the sipunculan and annelid Hr and myoHr sequences. Annelid myoHrs and sipunculan Hrs were resolved as monophyletic groups. Conversely sipunculan myoHrs did not form a clade. The Hrs having an octameric quaternary structure were resolved as a monophyletic group. The octameric cluster includes the Hr sequences of G. v. vulgaris, Themiste zostericola, Themiste discriptum, and Phascolopsis gouldii. Siphonosoma cumanense Hr, which has a trimeric quaternary structure, assumes a sister group position of the octameric clade. The S. nudus Hrs, having a quaternary structure that is not well resolved, assume an isolate position within the Hrs clade. Likelihood-based analyses reveal that purifying selection mainly characterized the evolution of Hr and myoHr. We suggest that starting from a common gene ancestor, two distinct quaternary structures evolved in the sipunculan Hrs and this differentiation was probably favored by the acquisition of distinct physiological advantages.

Oxymyohemerythrin: discriminating between O 2 release and autoxidation

Myohemerythrin (Mhr) is a non-heme iron O 2 carrier (with two irons in the active site) that is typically found in the retractor muscle of marine ‘peanut’ worms. OxyMhr may either release O 2, or undergo an autoxidation reaction in which hydrogen peroxide is released and diferric metMhr is produced. The autoxidation reaction can also be promoted by the addition of certain anions to Mhr solutions. This work, using recombinant Themiste zostericola Mhrs, contrasts the results of environmental effects on these reactions. For the O 2 release reaction, Δ V ‡(21.5°C)=+28±3 cm 3 mol −1, Δ H ‡(1 atm)=+22±1 kcal mol −1, and Δ S ‡(1 atm)=+ 28±4 eu. The autoxidation reaction (pH 8.0, 21.5°C, 1 atm) displays different kinetic parameters: Δ V ‡=−8±2 cm 3 mol −1, Δ H ‡=+24.1±0.7 kcal mol −1, and Δ S ‡=+1±1 eu. Autoxidation in the presence of sodium azide is orders of magnitude faster than solvolytic autoxidation. The Δ V ‡ parameters for azide anation and azide-assisted autoxidation reaction are +15±2 and +59±2 cm 3 mol −1, respectively, indicating that the rate-limiting steps for the Mhr autoxidation and anation reactions (including O 2 uptake) are not associated with ligand binding to the Fe2 center. The L103V and L103N oxyMhr mutants autoxidize ≈10 3–10 5 times faster than the wild-type protein, emphasizing the importance of leucine-103, which may function as a protein ‘gate’ in stabilizing bound dioxygen.

Structures of wild-type chloromet and L103N hydroxomet Themiste zostericola myohemerythrins at 1.8 A resolution.

Myohemerythrin (Mhr) is a nonheme iron oxygen carrier found in the retractor muscles of marine "peanut" worms. The X-ray crystal structures of two recombinant Themiste zostericola Mhrs are reported to a resolution of 1.8 A. Surprisingly, the met wild-type structure (R = 17.8%) was found to contain chloride bound to Fe2, while coordinated hydroxide was found in the met L103N structure (R = 18.3%). An internal water molecule was also found distal to the Fe-O-Fe center of the mutant protein, forming hydrogen bonds with the coordinated hydroxide and the OD1 atom of Asn-103. This finding is consistent with the kinetic and spectroscopic results reported for the L103N mutant Mhr [Raner, G. M., Martins, L. J., & Ellis, W. R., Jr. (1997) Biochemistry 36, 7037-7043]. Possible roles for the side chain of residue 103 (Leu in wild-type Mhr) in gating ligand binding are also discussed.

Functional role of leucine-103 in myohemerythrin.

Hemerythrins (Hrs) and myohemerythrins (Mhrs) are nonheme iron proteins that function as O2 carriers in four marine invertebrate phyla. Available amino acid sequences and X-ray structures indicate that a conserved leucine, residue 103 in the Themiste zostericola Mhr sequence, occupies a site distal to the Fe-O-Fe center. The side-chain methyl groups of the analogous leucine in Themiste dyscrita oxyHr are in van der Waals contact with bound O2 in the X-ray crystal structure, and this residue may therefore play a role in stabilizing bound dioxygen with respect to autoxidation. In order to test this hypothesis, the gene for T. zostericola Mhr was synthesized and expressed in Escherichia coli. Two mutant Mhrs, L103V and L103N, were also prepared. Optical spectra and kinetics data for these three proteins are presented. Importantly, neither mutant forms a stable oxy adduct; instead, rapid autoxidation results in formation of the corresponding met forms. In addition, the L103N Mhr displays unusually rapid reduction kinetics, suggesting that the amide functionality of Asn-103 destabilizes most bound ligands and additionally promotes rapid semi-metR <==> semi-metO isomerization.

Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C terminus of myohemerythrin.

In order to examine the effects of chain length on the propensity of short peptides to form helix-like structures in aqueous solution, we have studied a peptide of 31 residues consisting of the C-terminal sequence (residues 88-118) of the four-helix bundle protein myohemerythrin from Themiste zostericola. This peptide, termed MDC, represents the final two elements of secondary structure in the protein, the D-helix and the C-terminal loop sequence, together with a five-residue sequence at the N terminus corresponding to the linker between the C- and D-helices. An N-capping sequence, VDAKNV, immediately precedes the D-helix sequence, and a C-capping sequence, VNHIKGT, corresponding to the alphaL termination motif, occurs at the C-terminal end. The effect of replacement of a cysteine residue in the middle of the sequence with an alanine was explored by the comparison of the MDC peptide and a 16-residue peptide representing the sequence of the D-helix alone, both containing the change Cys99Ala. Significant changes in the NMR and CD spectra were seen for both peptides compared to the wild-type sequence. A comparison of the fluorescence spectra of the wild-type and Cys99Ala peptides indicated that a specific interaction between the side chains of Cys 99 and Trp 102 acts to quench the fluorescence of the tryptophan ring and probably contributes a component that distorts the CD spectrum of the wild-type peptide at approximately 220-235 nm. The effect of an increase in the length of the peptide, with the incorporation of capping sequences derived from the native sequence, was explored by NMR and CD spectroscopy of the 31-residue and 16-residue peptides in aqueous solution and in TFE/water mixtures. Evidence for the formation of a significant population of helical conformers in the region of the MDC peptide corresponding to the D-helix was observed in aqueous solution using CD and NMR spectroscopy. The C-terminal 10 residues of the MDC peptide behave in solution in a manner identical to that of a 10-residue peptide with the same sequence; a highly specific local interaction between an aromatic ring and a glycine amide proton appears to be retained in the longer peptide. Upon addition of trifluoroethanol (TFE), significant shifts are observed in a number of resonances in the NMR spectrum, and both chemical shifts and NOEs provide evidence for a higher population of helix in the D-helix region of the peptide in TFE. However, TFE is unable to promote the propagation of helix beyond the N-cap or alphaL termination motifs, and the specific local interaction observed in the C-terminal sequence is retained in TFE. The CD spectrum in TFE shows an increase in the proportion of helix, to an overall maximum of approximately 55% helix at 50% v/v TFE, corresponding to approximately 100% helix in the D-helix sequence of the peptide, since the N and C termini of the MDC peptide are not helical according to the NMR spectra. The high proportion of helix observed in the D-helix sequence of the longer MDC peptide demonstrates that the presence of intact capping sequences can constrain the peptide conformational ensemble to resemble that seen in the native protein. A compendium of results from this and previous peptide studies has also led to a novel observation, the existence of a correlation between the amide proton chemical shift and temperature coefficient.

Uptake and Release of O2 by Myohemerythrin. Evidence for Different Rate-Determining Steps and a Caveat

The oxy adducts of hemoglobins and hemerythrins are photosensitive, enabling the study of O{sub 2} recombination with deoxy forms produced by laser flash photolysis. Results to date have been interpreted in terms of consecutive O{sub 2} recombination equilibria for members of both protein families, indicating that O{sub 2} passage through these protein matrices involves multiple barriers. In relating rate and equilibrium data at physiological temperatures for these proteins, it has been tacitly assumed by many workers that K{sub eq}=k{sub on}/k{sub off}. In this communication, we report results for Themiste zostericola myohemerythrin (Mhr) and demonstrate that this assumption is not valid. Mhr binds dioxygen by reducing it to hydroperoxide; concomitantly, diferrous deoxyMhr is oxidized to the diferric oxy form. The (hydro)peroxo {yields} Fe{sup III} charge-transfer transition, centered at 500 nm, of oxyMhr was used to monitor the kinetics of O{sub 2} uptake and release. Rapid mixings of oxyMhr with sodium dithionite was used to scavenge O{sub 2} in solution, initiating O{sub 2} release from the protein. Rate constants for this reaction as a function of temperature and pressure are plotted and summarized. These results point to a rate-determining step that occurs prior to Fe-O bond formation. 14 refs., 3 figs., 1more » tab.« less

Amino Acid Sequence of the Hemerythrin α Subunit from Lingula unguis

The amino acid sequence of the alpha subunit of the allosteric hemerythrin from Lingula unguis was determined. It consists of 117 amino acid residues. Compared with other non-allosteric hemerythrins consisting of identical subunits of 113 amino acid residues, this protein has the deletion of the N-terminal amino acid and the insertion of five amino acids in the same region as in the case of the monomeric myoerythrin from Themiste zostericola. As the amino acid sequence of the beta subunit has also been determined [Yano, H., Satake, K., Ueno, Y., & Tsugita, A. Protein Sequence and Data Analysis, in press], the complete sequence analysis of an allosteric hemerythrin has been accomplished for the first time. The difference in the octameric structures of allosteric and non-allosteric hemerythrins are discussed.

Primary structure of myohemerythrin from the annelid Nereis diversicolor

The metal-free form of Nereis diversicolor myohemerythrin was purified from whole animal extracts by trichloroacetic acid precipitation and ion exchange chromatography. The amino acid sequence of myohemerythrin has been determined. The protein is composed of 120 residues, possesses an unblocked N-terminus and is devoid of cysteine residues. It bears 62% sequence identity with Themiste zostericola myohemerythrin, the only other member of this subfamily sequenced to date. Within the family of hemerythrins, homology is particularly high in the segments involved in the binding of the two iron atoms and in the β-turn-rich N-terminal segment.

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin.

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.

The interaction of hemerythrin with sodium dodecyl sulfate and the release of iron from the product with desteferrioxamine B

The interactions with sodium dodecyl sulfate (SDS) of methemerythrin, the anionic derivatives and oxyhemerythrin from Phascolopsis gouldii have been examined at 25°C, I = 0.5 M and pH 6.3 and 7.8. Absorbance changes in the 350–500 nm range were used to monitor the rates. The denaturation is slow ( k = 10 −2–10 −3 s −1) and only slightly dependent on SDS concentration. Perchlorate is a very effective inhibitor of the SDS reaction with methemerythrin, and it is concluded that rapid binding of SDS near to the cysteine-50 site is an essential to unfolding. Myohemerythrin (from Themiste zostericola) and the monomeric N-ethylmaleimide derivatives of methemerythrin from P. gouldii and T. zostericola, in contrast, react rapidly with SDS. The products from denaturing of all proteins appear similar, having reduced α-helix content, very small absorbance in the 350–500 nm region and loss of anion or oxygen binding capacity. They do, however, retain the two irons, which can readily be removed with desferrioxamine B.

The kinetics of oxidation of hemerythrin species—linear free energy relationships

The second-order rate constants (M −1sec −1, 25°C, pH 8.2, I = 0.15 M) for the oxidation to (semi-met) 0of deoxyhemerythrin from Phascolopsis gouldii (P.g.) and Themiste zostericola (T.z.) have been determined for Fe(CN) 5(4-NH 2py) 2− (3.6 × 10 4 T.z.,2.8 × 10 2P.g.),Fe(CN) 5NH 3 2−(2.4 × 10 4 T.z.), Fe(CN) 6 3− (1.0 × 10 5 T.z.,1.4 × 10 2P.g.), Fe(CN) 5PPh 3 2− (7.3 × 10 5T.z.), and Fe(CN) 4dipy- (~6 × 10 6 T.z.,7.5 × 10 4 P.g.). Corresponding rate constants for the oxidation of (semi-met) R to met are: Fe(CN) 5(4-NH 2py) 2− (1.2 × 10 3 P.g.), Fe(CN) 6 3− (3.4 × 10 5 T.z., 4.5 × 10 Fe(CN) 5PPh 3 2− (4.4 × 10 4P.g.), Fe(CN) 4dipy − (1.7 × 10 5P.g.), and Coterpy 2 3+ (5.1 P.g.) The rates of oxidation of deoxy- and (semi-met) R myohemetythrin by Fe(CN) 6 3− were too rapid for stopped-flow measurement. The Marcus relationship for cross-reactions was successfully applied to these data.

Reduction potentials of various hemerythrin oxidation states

The reduction potentials of the couple methemerythrin/(semi-met) r have been determined spectrally, using 2,6-dichlorophenolindophenol as a redox partner, for monomer and octamer from Themiste zostericola and for octamer protein from Phascolopsis gouldii. The reduction potentials of the couple (semi-met) o/deoxy have been determined spectrally, using Fe(III) cyanocomplexes, for monomer and octamer protein from Themiste zostericola. The values of other potentials, (met/(semi-met) o and (semi-met) r /deoxy), have thus been deduced as well as equilibrium constants for the (semi-met) r α (semi-met) o conformational changes for hemerythrins from Themiste zostericola.

Kinetics of the equilibration of oxygen with monomeric and octameric hemerythrin from Themiste zostericola

Single relaxations for the equilibration of O 2 with monomeric and octameric deoxy forms of hemerythrin from Themiste zostericola have been observed at 25°C using the temperature-jump technique. At 25°C, pH 8.2 (Tris/H 2SO 4 and I = 0.10 M (Na 2SO 4), formation rate constants k on are 7.8 · 10 7 M −1 · s −1 and 7.5 · 10 6 M −1 s −1, respectively. The procedure used does not give a precise measure (small intercepts) of dissociation rate constants, k off . These were determined instead by the stopped-flow method using dithionite to induce dissociation of the oxy protein. Values of k off for the monomer (3.1 · 10 2 s −1) and octamer (82 s −1), in association with k on values, lead to equilibrium constants for the formation of oxyhemerythrin of 2.5 · 10 5 M −1 and 0.9 · 10 5 M −1, respectively, at 25°C, pH 8.2 and I = 0.10 M (Na 2SO 4). These latter are in reasonable agreement with values (1.5 10 5 M −1 and 1.3 · 10 5 M −1) determined spectrally on the equilibrated solutions. Using the octameric protein, it was shown that replacement of SO 4 2− by ClO 4 − or Cl − ions (at a constant I = 0.10 M ) led to an approximately 2-fold enhancement of k on but had little effect on k off . The addition of Ca 2+ or Mg 2+ ions (0.01 M), with or without 0.50 M NaCl, also gives up to 4-fold increases in k on , but unchanged k off values. Oxygen pulse experiments on the octamer show no effect on k off of the degree of oxygenation of the protein. A comparison is made with similar data for hemoglobin, myoglobin and hemocyanin.

EPR spectroscopy of semi-methemerythrin

EPR spectra of semi-met forms of octameric hemerythrin from Themiste zostericola, prepared by one electron reduction of methemerythrin or by one electron oxidation of deoxyhemerythrin, have been visualized at liquid helium temperatures. The spectrum of that prepared by one electron reduction has principal g-values of 1.96 ± 0.01, 1.88 ± 0.01, and 1.67± 0.02 while that obtained by one electron oxidation has g = 1.95 ± 0.01, 1.72 ±0.01, and 1.68 ± 0.02. The amplitude of either spectrum decreases with time on incubation at room temperature according to a first order rate with t 1 2 = 5–8 min , apparently because of an intramolecular disproportionation. Similar EPR spectra have been obtained with semi-metmyohemerythrin of T. zostericola and with the octameric semi-met form of Phascolopsis gouldii. However, these forms disproportionate to a much lesser degree. The azide adduct of the octameric semi-met form of T. zostericola has g-values of 1.94 ± 0.01, 1.85 ± 0.01, and 1.57 ± 0.02. Its EPR spectrum differs somewhat from those of the azide adducts of the octamer of P. gouldii and the monomer of T. zostericola although all are resistant to disproportionation. Methemerythrin and deoxyhemerythrin have no EPR spectra even at liquid helium temperature.

Role of mixed oxidation states in the oxidation of hemerythrin species by ferricyanide ion.

The oxidation of deoxy- and oxyhemerythrin to methemerythrin by Fe(CN)6(3-) has been examined. The first step produces (semi-met)O, a half-oxidized form of hemerythrin which has different spectral and kinetic properties from (semi-met)R which is produced by one-electron reduction of methemerythrin. Further oxidation of (semi-met)O to methemerythrin usually occurs only indirectly via disproportionation of (semi-met)O to met and deoxy forms within the octameric framework. Oxidation of (semi-met)R by Fe(CN)6(3-) is a direct second-order reaction. (Semi-met)O is reduced rapidly by dithionite to deoxy, whereas that of (semi-met)R is a slow biphasic process. The oxidation of oxyhemerythrin occurs via the deoxy species, little, if any, reactivity being attributable to the oxy form. The oxidation of the azide adduct of (semi-met)R is biphasic, in which in step one N3(-) is removed during the oxidation to methemerythrin, which in the second step recombines with N3(-). Rate parameters for all these processes at pH 8.2 and 6.3 for protein from Phascolopsis gouldii and Themiste zostericola have been obtained. The implications of these findings to hemerythrin chemistry are discussed.

Pseudosymmetry in the structure of myohemerythrin.

The three-dimensional structure of the protein myohemerythrin from retractor muscles of the sipunculan worn Themiste zostericola has been explored for the existance of approximately symmetry operators that locally interrelate portions of the molecule. First, the electron denisty distribution at 5.5 A resolution was examined. A local 2-fold axis that transposes the C-D helix pair into the A-B helix pair was found and refined by the method of least squares. The match in electron densities for a pure 2-fold rotation had a correlation coefficient of 0.56. Next, a comprehensive search was made for rotational symmetry in the Patterson function of an isolated molecule. The rotation function based on data to 6 A spacings showed a major peak, 72% of the self-peak height, that confirmed the result from electron density correlations. In addition, a pattern of lower level peaks revealed approximate point group symmetry as high as D4. Finally, the amino acid sequence has been inspected for evidence of a repeated structure. The level of amino acid identities between positions in the A-B and C-D helix pairs is 28%. Several factors are discussed to suggest that this homology, although low, is nonetheless significant.