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The complete amino-acid sequences of the alpha- and beta-subunits of hemerythrin from the brachiopod Lingula reevii have been determined. These subunits are found in equal proportions in coelomic hemerythrocytes and form an octamer, presumably with an alpha 4 beta 4 composition. Both subunits were found to consist of 117 residues and to show 65% sequence identity to each other. Sequences of the alpha- and beta-subunits of L. reevii hemerythrin are closely related to their counterparts in hemerythrin from the related brachiopod, Lingula unguis, showing 95% and 87% sequence identities, respectively. Sequence alignments show that 25 residues in the lingulid hemerythrin subunits are identical to those found in other hemerythrins and myohemerythrins. These conserved residues include those known to provide iron ligands. However, these comparisons also indicate that the lingulid hemerythrin sequences are distinct from those of the sipunculid and annelid hemerythrins and myohemerythrins.
The crystallographic structure analyses of deoxy and oxy hemerythrin have been carried out at 2.0 A resolution to extend the low resolution views of the physiological forms of this oxygen-binding protein. Restrained least-squares refinement has produced molecular models giving R-values of 16.8% for deoxy (41,064 reflections from 10 A to 2.0 A) and 17.3% for oxy hemerythrin (40,413 reflections from 10.0 A to 2.0 A). The protein structure in each derivative is very similar to that of myohemerythrin and the various met forms of hemerythrin. The binuclear complex in each derivative retains an oxygen atom bridging the two iron atoms, but the bond lengths found in deoxy hemerythrin support the idea that, in that form, the bridge is protonated, i.e. the bridging group is a hydroxyl. Dioxygen binds to the pentaco-ordinate iron atom in deoxy hemerythrin in the conversion to oxy hemerythrin. The interatomic distances are consistent with the proposed mechanism where the proton from the bridging group is transferred to the bound dioxygen, stabilizing it in the peroxo oxidation state by forming a hydrogen bond between the peroxy group and the bridging oxygen atom.
The crystallographic refinement of met and azidomet hemerythrin has been carried out at 1.66 A resolution in an attempt to characterize precisely the binuclear iron center in this protein. Restrained least-squares refinement has produced molecular models giving R-values of 18.9% for met (65,683 reflections from 10 A to 1.66 A) and 17.6% for azidomet hemerythrin (68,747 reflections from 10.0 A to 1.66 A). The protein structure in each derivative is very similar to that of myohemerythrin. The mu-oxo bridged iron center differs between the two forms. The complex in met hemerythrin is asymmetric with the bridging oxygen closer to one of the iron atoms while the complex in azidomet hemerythrin is symmetric. After investigations of the effects of correlation in the refinement, we believe this difference between the two complexes is associated with chemical differences and is not a refinement artefact.
Hemerythrin from coelomic cells of Phascolosoma lurco (syn. P. arcuatum) was isolated by gel filtration as two components, hemerythrin-I (25%) and hemerythrin-II (75%). The Mössbauer spectrum of oxyhemerythrin-II consisted of two pairs of lines of the same isomer shift (0.5 mm s(-1) corresponding to Fe(III) but different quadrupole splitting (1.01 and 2.02 mm s(-1). Application of a 2.5-T magnetic field at 4.2 K caused no significant spectral broadening. The 2FE.O2 binding site thus contains two nonequivalent high-spin Fe(III) ions that are antiferromagnetically coupled. The Mössbauer spectra of the minor component, hemerythrin-I, indicated an identical binding site. On deoxygenation, the spectrum was dominated by a simple quadrupole split doublet corresponding to Fe(II), indicating that the binding site in this derivative contains two identical Fe(II) ions that interact only weakly, if at all. The Mössbauer spectra of azidohemerythrin-II indicated that this derivative also contains a pair of antiferromagnetically coupled Fe(III) ions with the same isomer shift (0.5 mm s(-1)) but quadrupole splittings (1.40 and 1.96 mm s(-1)) that are not identical with those in oxyhemerythrin.
The solution structures of the binuclear Mn centers in arginase, Mn catalase, and the Mn-substituted forms of the Fe enzymes ribonucleotide reductase and hemerythrin have been determined using X-ray absorption spectroscopy (XAS). X-ray absorption near edge structure (XANES) spectra for these proteins were compared to those obtained for Mn(II) models. The Mn model spectra show an inverse correlation between the XANES peak maximum and the root-mean-square (RMS) deviation in metal-ligand bond lengths. For these complexes, the XANES maxima appear to be more effective than the 1s --> 3d areas as an indicator of metal-site symmetry. Arginase and Mn-substituted ribonucleotide reductase have symmetric nearest neighbor environments with low RMS deviation in bond length, while Mn catalase and Mn-substituted hemerythrin appear to have a larger RMS bond length deviation. The 1s --> 3d areas for arginase and Mn-substituted ribonucleotide reductase are consistent with six coordinate Mn, while the 1s --> 3d areas for Mn catalase and Mn-substituted hemerythrin are larger, suggesting that one or both of the Mn ions are five-coordinate in these proteins. Extended x-ray absorption fine structure (EXAFS) spectra were used to determine the Mn2 core structure for the four proteins. In order to quantitate the number of histidine residues bound to the Mn2 centers, EXAFS data for the crystallographically characterized model hexakis-imidazole Mn(II) dichloride tetrahydrate were used to calibrate the Mn-imidazole multiple scattering interactions. These calibrated parameters allowed the outer shell EXAFS to be fit to give a lower limit on the number of bound histidine residues. The EXAFS spectra for Mn-substituted ribonucleotide reductase and arginase are nearly identical, with symmetric Mn-nearest neighbor environments and outer shell scattering consistent with a lower limit of one histidine per Mn2 core. In contrast, the EXAFS data for Mn catalase and Mn-substituted hemerythrin show two distinct Mn-nearest neighbor shells, modeled as Mn-O at ca. 2.1 A and Mn-N at ca. 2.3 A, and outer shell carbon scattering consistent with a lower limit of ca. 2-3 His residues per Mn2 core. Only Mn catalase shows clear evidence for Mn...Mn scattering. The observed Mn...Mn distance is 3.53 A, which is significantly longer than the approximately 3.3 A distances that are typically observed for Mn(II)2 cores with two single atom bridges, but which is typical of the distances seen in Mn(II)2 cores having one single atom bridge (e.g., aqua or hydroxo) together with one or two carboxylate bridges. The absence of EXAFS-detectable Mn...Mn interactions for the other three proteins suggests either that there are no single atom bridges in these cases or that the Mn...Mn interactions are more disordered.
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.
Electron-transfer reactions of myoglobin, hemocyanin and hemerythrin with the inorganic complexes [Fe(CN)6]3- (oxidant) and [Co(sep)]2+ (reductant) are considered. Rate constants kFe (25 degrees C) have been determined for the [Fe(CN)6]3- (410 mV) oxidation of horse deoxyMb, I = 0.100 M (NaCl). From the decrease in kFe over the range pH 5.5 to 9.0 a pKa of less than 6.2 is obtained, most likely due to the involvement of the heme propionate(s). At the higher pH values the rate constant is 1.2 x 10(6) M-1 s-1. Rate constants kCo (25 degrees C) for the [Co(sep)]2+ (-260 mV) reduction of metMb are also pH-dependent, pKa = 8.82, corresponding to acid dissociation of the H2O axially coordinated to the Fe(III). The rate constant for the aqua-met form is 2.8 x 10(3) M-1 s-1 at pH values less than 7.0. In contrast, no reaction is observed for the deoxy and met forms of P. interruptus hemocyanin monomer subunit a with the same two complexes (k less than 10(2) M-1 s-1). Comparisons are made with rate constants for hemerythrin, also as the monomer, which have been determined previously. Rate constants for the reactions of deoxy forms with the neutral small molecules, here O2 and H2O2, are also considered. Whereas the reactions of [Fe(CN)6]3- and [Co(sep)]2+ are at the protein surface, those of O2 and H2O2 are at the active site. Hemocyanin with the more buried (approximately 20 A) active site compared with myoglobin (3.8 A) and hemerythrin (6.3 A), does not readily undergo electron transfer with reagents at the surface. However, with the small molecules O2 and H2O2 penetration of the surrounding peptide occurs, with reaction at the active site. Rate constants for the three proteins are now of similar magnitude, and in the range (2.3-7.8) x 10(7) M-1 s-1 for O2, and 10.9 to 3600 M-1 s-1 for H2O2.
Resonance Raman, optical absorption, circular dichroic, and fluorescence emission spectroscopy of hemerythrins from four species of sipunculids (Phascolopsis gouldii, Phascolosoma agassizii, Themiste dyscritium, and Themiste pyroides) reveals no major differences in their active site or tertiary structures. This precludes any change in iron ligands or coodination geometry and makes it unlikely that the active-site structures of P. gouldii and T. dyscritum hemerythrins could be as disparate as indicated by present crystallographic interpretations (Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 349; Klotz, I. M., Klippenstein, G. L., and Hendrickson, W. A. (1976), Science 192, 335). Resonance Raman enhancement profiles of the stretching modes involving coordinated dioxygen maximize with excitation at approximately 525 nm, and correspond to the circular dichroic (CD) transition at approximately 520 nm. For coordinated azide modes in metazidohemerythrins these profiles maximize with excitation at approximately 505 nm corresponding to the 500-nm CD transition. Hemerythrins also possess another resonance Raman peak at approximately 510 cm-1 which show maximum intensity enhancement at approximately 530 nm and this vibration is most likely associated with a permanent iron ligand.
A leucine residue, Leu-98, lines the O(2)-binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O(2) association rate constants within a factor of 3 of the wild type value. Similarly, the O(2) dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O(2) dissociation rate constant and a large D(2)O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O(2). Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X = Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O(2), but is evident for solvent.
The amino acid sequence of the beta chain of hemerythrin from Lingula unguis was determined. The sequence consists of 117 amino acid residues. The sequence shares 25 amino acid residues in common with other known hemerythrin sequences, including myohemerythrin, with which it shares an insertion of 5 amino acids. This is the first complete sequence from a heteropolymeric hemerythrin.
The first enzyme of the common aromatic biosynthetic pathway in Escherichia coli, the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, contains iron as an integral part of the polypeptide chain, and the enzyme shows an absorption maximum around 350 nm (McCandliss, R.J., and Herrmann, K.M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4810-4813). These two properties are also found in hemerythrin, the oxygen carrier of certain marine invertebrates. The amino acid sequence of residues 10 to 18 of the enzyme from E. coli, His-Ile-Thr-Asp-Glu-Gln-Val-Leu-Met, is highly homologous to the sequence of residues 54 to 62 of hemerythrin from Phascolopsis gouldii, His-Phe-Leu-Asn-Glu-Gln-Val-Leu-Met. His54 and Glu58 of hemerythrin have previously been identified through x-ray and protein sequence analysis as iron ligands. We suggest that residues 10 to 18 of the E. coli enzyme represent part of the iron binding fold in this protein, and that His10 and Glu14 are iron ligands.
Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pk of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin. Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association. Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron.
A recent report from this laboratory (Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. and Debrunner, P.G. (1991) Biochemistry 30, 583-589) described a procedure for reconstitution of a functional di-iron site in the octameric, non-heme iron O2-carrying protein, hemerythrin by addition of ferrous salts to apoprotein, followed by slow dilution of the denaturant. Although the resulting protein contained its full complement of iron, i.e., 2 Fe per subunit, about 30% of the iron was found to remain ferrous under ambient O2, i.e., this iron was incapable of forming an O2 adduct. In this report a method is described for obtaining essentially fully functional hemerythrin by passage of the freshly reconstituted protein through an [oxy/30% non-functional----met----deoxy----oxy redox cycle. UV/vis absorption and 57Fe Mössbauer spectroscopies show that little or no non-functional iron remains in the reconstituted oxyhemerythrin after the redox cycle. Quantitations of protein and diiron sites show that, during the first step of the redox cycle, the non-functional iron is converted to a form that is spectroscopically indistinguishable from that of native methemerythrin. Far-UV circular dichroism shows that the secondary structure of this reconstituted methemerythrin is essentially identical to that of native protein. Non-denaturing polyacrylamide gel electrophoresis shows that the size and charge of the native and reconstituted proteins before and after redox cycling are essentially identical. These results indicate that the non-functional iron is converted to a functional form by the redox cycling, and that the key step in this conversion is the [oxy/30% non-functional]----met transformation.
Exposure of methemerythrin (metHr) to S2- under anaerobic conditions results in a one-electron reduction to the semi-met level and replacement of the mu-oxo bridge between the irons with a single sulfide. The sulfide bridge is maintained upon ferricyanide oxidation of semi-metsulfide to metsulfide hemerythrin and upon subsequent dithionite or S2- reduction back to the semi-met level. Chemical analyses show that metsulfideHr contains one S2- per two Fe. The single quadrupole doublet (delta = 0.50 mm/s; delta Eq = 0.99 mm/s) in the Mössbauer spectrum is consistent with a bridging sulfide geometry. The optical and resonance Raman spectra of metsulfideHr are reminiscent of the [2Fe-2S] iron-sulfur proteins. The optical spectrum exhibits multiple S2----Fe(III) charge-transfer transitions between 400 and 600 nm. The resonance Raman spectrum reveals a series of overtones and combinations of the 431-cm-1 Fe-S-Fe symmetric vibration and the 327-cm-1 asymmetric vibration. The relative energies of the symmetric and asymmetric modes are characteristic of a sulfur-bridged system with a bridge angle of approximately 80 degrees. MetsulfideHr decomposes over several hours in air and over several days in the absence of O2 to metHr and semi-metsulfideHr, respectively. Unlike metHr and semi-metHr, neither the metsulfide nor the semi-metsulfide derivatives form stable adducts with anions such as azide or cyanide. Sulfide bridging confers new properties on the binuclear iron center that are of interest to an understanding of the chemistry of hemerythrin and also of the [2Fe-2S] iron-sulfur proteins.
The reactions of hemerythrin from Phascolopsis gouldii with the specific sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoate), 2,2'-dithiodipyridine, and 4,4'-dithiodipyridine were studied at 25 degrees C. Spectrophotometric measurements showed that 1 mol of disulfide reacted per protein subunit consistent with a single cysteine at residue 50. Reaction leads to dissociation of the octameric structure of the native protein to monomers. The first-order rate constants at 25 degrees C and pH 9.0 for reactions of methemerythrin [(1.5 +/- 0.3) X 10(-3) s-1] and metazidohemerythrin [(4.0 +/- 0.3) X 10(-3) s-1] are independent of both the concentration and the nature of the disulfide. The reactions of methemerythrin are strongly inhibited by ClO4-ion, which however has no effect on the rates of those of metazidohemerythrin. The first-order kinetic behavior is ascribed to a conformational change involving the protein controlling the reaction, and this slow change appears to dominate a number of the reactions of hemerythrin.
Extensive X-ray absorption fine structure measurements and analysis have been made on azidomet- and methemerythrin and on the native forms of oxy- and deoxyhemerythrin. Due to the availability of models that have been synthesized to mimic the active site of hemerythrin, it was possible to make a thorough assessment of the various errors in the structural parameters determined by the analysis. It is found that the largest source of error is the lack of complete transferability of amplitude and phase between the standards and hemerythrin. This is of particular importance in distinguishing the contributions of the second-shell low-Z atoms and, thus, has a substantial influence on the determination of the iron-iron distance. The internal consistencies of the various checks and a new formulation of error analysis for the structural parameters give us confidence in the structure determined for the active site. The main result is that as O2 is released from oxyhemerythrin, the mu-oxo bridge between the two iron atoms in the active site with an Fe-O distance of 1.8 A converts to a mu-hydroxo bridge in deoxyhemerythrin, expanding the Fe-O distance to 2.0 A. The Fe-Fe distance expands proportionally from 3.24 A in oxyhemerythrin to 3.57 A in deoxyhemerythrin so as to keep the Fe-O-Fe bridging angle approximately constant. These conclusions provide experimental support for the structures of oxy- and deoxyhemerythrin proposed previously on the basis of spectroscopic and preliminary X-ray crystallographic data.