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The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.

A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

The first demonstrated example of a regulatory function for a bacterial hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O(2) and undergo autoxidation. The amino acid sequence encoded by the VC1216 gene from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial diguanylate cyclases (DGCs) that catalyze formation of cyclic di-(3',5')-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon being exposed to air. Vc Bhr-DGC showed approximately 10 times higher DGC activity in the diferrous than in the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in V. cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low-dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity.

Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO). Recently, we showed that FMN-free diferrous FDP from Thermotoga maritima exposed to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP(NO)) that can react further with NO to form N(2)O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040-7049]. Here we report resonance Raman and low-temperature photolysis FTIR data that better define the structure of this diiron-mononitrosyl complex. We first validate this approach using the stable diiron-mononitrosyl complex of hemerythrin, Hr(NO), for which we observe a ν(NO) at 1658 cm(-1), the lowest ν(NO) ever reported for a nonheme {FeNO}(7) species. Both deflavo-FDP(NO) and the mononitrosyl adduct of the flavinated FPD (FDP(NO)) show ν(NO) at 1681 cm(-1), which is also unusually low. These results indicate that, in Hr(NO) and FDP(NO), the coordinated NO is exceptionally electron rich, more closely approaching the Fe(III)(NO(-)) resonance structure. In the case of Hr(NO), this polarization may be promoted by steric enforcement of an unusually small FeNO angle, while in FDP(NO), the Fe(III)(NO(-)) structure may be due to a semibridging electrostatic interaction with the second Fe(II) ion. In Hr(NO), accessibility and steric constraints prevent further reaction of the diiron-mononitrosyl complex with NO, whereas in FDP(NO) the increased nucleophilicity of the nitrosyl group may promote attack by a second NO to produce N(2)O. This latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol. Inorg. Chem. 2007, 12, 79-89]. Published vibrational data on bioengineered models of denitrifying heme-nonheme NO reductases [Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939-5947 ] support a similar mode of activation of a heme {FeNO}(7) species by the nearby nonheme Fe(II)., (© 2012 American Chemical Society)

Hemerythrin is proposed as an alternative to hemoglobin-based blood substitutes. In contrast to hemoglobin, hemerythrin exhibits negligible reactivity towards oxidative and nitrosative stress agents (peroxide, nitric oxide, nitrite). Protocols for attachment of polyethylene glycol and glutaraldehyde cross-linking of Hr are described. These derivatizations appear to have favorable effects on O(2) affinity and autoxidation rates for use in blood substitutes. Based on lessons learned from hemoglobin-based blood substitutes, these derivatizations should also help limit extravasation and antigenicity of a hemerythrin-based blood substitute.

An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H2O2 and O2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H2O2 oxidoreductase) and NADH oxidase (NADH : O2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H2O2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H2O2 and O2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.

The methyl-accepting chemotaxis protein, DcrH, from the anaerobic sulfate-reducing bacterium, Desulfovibrio vulgaris (Hildenborough), has a hemerythrin-like domain, DcrH-Hr, at its C terminus. DcrH-Hr was previously shown to contain a diiron site that binds O2, suggesting an O2-sensing function. X-ray crystal structures of diferric (met-), azido-diferric (azidomet-), and diferrous (deoxy-) DcrH-Hr reveal a "substrate tunnel" distinct from that in invertebrate hemerythrins. This tunnel is proposed to facilitate the rapid autoxidation of oxy-DcrH-Hr and suggests that sensing is triggered by O2 binding and subsequent oxidation of the diferrous active site. The N-terminal loop of DcrH-Hr is highly ordered in both met- and azidomet-DcrH-Hr but is disordered in deoxy-DcrH-Hr. These redox-dependent conformational differences presumably transduce the sensory signal of DcrH-Hr to the neighboring methylation domain in the full-length receptor. Given the putative cytoplasmic localization of its Hr-like O2-sensing domain, DcrH is proposed to serve a role in negative aerotaxis (anaerotaxis).

High-resolution crystal structures of Desulfovibrio vulgaris nigerythrin (DvNgr), a member of the rubrerythrin (Rbr) family, demonstrate an approximately 2-A movement of one iron (Fe1) of the diiron site from a carboxylate to a histidine ligand upon conversion of the mixed-valent ([Fe2(II),Fe1(III)]) to diferrous states, even at cryogenic temperatures. This Glu<-->His ligand "toggling" of one iron, which also occurs in DvRbr, thus, appears to be a characteristic feature of Rbr-type diiron sites. Unique features of DvNgr revealed by these structures include redox-induced flipping of a peptide carbonyl that reversibly forms a hydrogen bond to the histidine ligand to Fe1 of the diiron site, an intra-subunit proximal orientation of the rubredoxin-(Rub)-like and diiron domains, and an electron transfer pathway consisting of six covalent and two hydrogen bonds connecting the Rub-like iron with Fe2 of the diiron site. This pathway can account for DvNgr's relatively rapid peroxidase turnover. The characteristic combination of iron sites together with the redox-dependent iron toggling between protein ligands can account for the selectivity of Rbrs for hydrogen peroxide over dioxygen.

Reported are the X-ray crystal structures of recombinant Phascolopsis gouldii methemerythrin (1.8-A resolution) and the structure of an O2-binding-pocket mutant, L98Y methemerythrin (2.1-A resolution). The L98Y hemerythrin (Hr) has a greatly enhanced O2 affinity, a slower O2 dissociation rate, a larger solvent deuterium isotope effect on this rate, and a greater resistance to autoxidation relative to the wild-type protein. The crystal structures show that the hydrophobic binding pocket of Hr can accommodate substitution of a leucyl by a tyrosyl side chain with relatively minor structural rearrangements. UV/vis and resonance Raman spectra show that in solution L98Y methemerythrin contains a mixture of two diiron site structures differing by the absence or presence of an Fe(III)-coordinated phenolate. However, in the crystal, only one L98Y diiron site structure is seen, in which the Y98 hydroxyl is not a ligand, but instead forms a hydrogen bond to a terminal hydroxo/aqua ligand to the nearest iron. Based on this crystal structure, we propose that in the oxy form of L98Y hemerythrin the non-polar nature of the binding pocket favors localization of the Y98 hydroxyl near the O2 binding site, where it can donate a hydrogen bond to the hydroperoxo ligand. The stabilizing Y98OH-O2H-interaction would account for all of the altered O2 binding properties of L98Y Hr listed above.

A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

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.

Hemerythrin (Hr) is an O(2)-carrying protein found in some marine invertebrates. A conserved sequence motif in all Hrs provides five histidine and two carboxylate ligands to an oxo-/hydroxo-bridged diiron active site, as well as a hydrophobic O(2) binding pocket. Database searches located a previously unrecognized Hr-like sequence motif at the 3' end of the gene, dcrH, from the anaerobic sulfate-reducing bacterium, Desulfovibrio (D.) vulgaris (Hildenborough). This gene encodes a putative methyl-accepting chemotaxis protein, DcrH. We have established by immunoblotting that a full-length DcrH, including the Hr-like domain, is expressed in D. vulgaris (Hildenborough). The C-terminal domain of DcrH, when expressed separately in recombinant form in Escherichia coli, was found to fold into a stable protein, DcrH-Hr. The UV-vis absorption and resonance Raman spectra of DcrH-Hr, and of its azide adduct, provide clear evidence for an oxo-bridged diiron(III) site very similar to that found in Hr. Based on UV-vis absorption spectra, exposure of the reduced (colorless, presumably diferrous) DcrH-Hr to air resulted in formation of an O(2) adduct also very similar to that of Hr. Unlike that of Hr, the O(2) adduct of DcrH-Hr autoxidized within a few minutes at room temperature. The O(2) binding pocket of DcrH-Hr appears to be larger than that of Hr. Given the air-sensitive nature of D. vulgaris and the putative chemotactic function of DcrH, one possible role for the Hr-like domain of DcrH is O(2)-sensing. DcrH-Hr is the first characterized example of a Hr-like protein from any microorganism.

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.

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp Sal1 fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe3+(SCys)4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.

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.

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.

A general method is described for substitution of Mn(II) and Co(II) into the diiron sites of hemerythrin and myohemerythrin. Characterizations of these metal-substituted proteins show that their structures closely resemble those of the native proteins. In particular, the four-helix bundle structure appears to be maintained. The apomyohemerythrin retains most of the native helix content but is considerably less stable to denaturation than are the metal-containing proteins. The relative affinities of M(II) for apohemerythrin--namely, Co greater than Fe greater than Mn--parallel the stabilities of the M2myohemerythrins to denaturation by guanidinium chloride. These results indicate that for myohemerythrin (i) the majority of the helical structure found in the native protein does not require incorporation of M(II) and (ii) stabilization of the native structure relative to the fully unfolded structure appears to be due predominantly to M(II)-protein interactions, at least for M = Fe and Co. Incorporation of M(II) also generates unfolding cooperativity in myohemerythrin. This cooperativity can be attributed to interhelical interactions, which are prevented in the apoprotein by solvation of the seven metal ligand residues. The results are consistent with a minimal model for folding/unfolding of myohemerythrin and hemerythrin subunits consisting of the sequential equilibria, N in equilibrium with I in equilibrium with D, between native, intermediate, and fully unfolded states, respectively. The properties of apomyohemerythrin make it a candidate for the intermediate state, I.

Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.

Reported are results on the subunit composition of octameric hemerythrin (Hr) from the brachiopod Lingula reevii. Unlike most other Hrs, L. reevii Hr shows cooperativity in O2 binding. Purified L. reevii Hr was found to consist of two different subunits in approximately equimolar proportions. These two subunits differ in molecular weight by approximately 1000. Amino acid sequence data for the first 24 residues of the two subunits, labeled alpha and beta, show 70% identity with each other. Comparisons to amino acid sequences of other Hrs show approximately 50% identity in the first 24 residues and that both the alpha and beta subunits of L. reevii Hr have one residue deleted at their amino termini. Very recently, one other Hr, that from the brachiopod Lingula unguis, was also shown to contain equimolar proportions of two different subunits [Satake, K., Yugi, M., Kamo, M., Kihara, H., & Tsugita, A. (1990) Protein Seq. Data Anal. 3, 1-5], and this Hr also shows cooperativity in O2 binding. An alpha 4 beta 4 octamer is, therefore, proposed to be a common feature of those Hrs that show such cooperativity. Likely arrangements of alpha and beta subunits within an alpha 4 beta 4 octamer having the same configuration of subunits as that in other octameric Hrs are proposed. The most probable arrangements can be readily derived from physically reasonable restrictions on the types of intersubunit interactions and on transmission of allosteric effects.

The first reconstitutions of functional diiron sites in the nonheme O2-carrying proteins hemerythrin (Hr) and myohemerythrin (myoHr) have been achieved. Both proteins are reconstituted under anaerobic conditions, and the procedure consists of (i) denaturation of the native met form with 6 M guanidinium chloride in the presence of sodium dithionite and 2,2'-dipyridyl, (ii) separation of the apoprotein from the other reagents and products, (iii) addition of an iron(II) stock solution to the apoprotein in the presence of 2-mercaptoethanol, and (iv) several cycles of slow dilution and reconcentration by ultrafiltration to remove excess reagents. Iron analyses indicate that the apoproteins have been essentially completely freed of iron and that reconstituted Hr contains its full complement of iron, i.e., approximately 2 Fe/subunit. Ferrous rather than ferric iron appears to be necessary for recovery of the native structures for both myoHr and Hr. In the case of Hr, reconstitution was successful only when iron(II) was added to apoHr prior to removal of denaturant. ApoHr is essentially insoluble at pH 7 in the absence of denaturants but remains soluble when denaturant is removed in the presence of ferrous iron, which leads to recovery of the octameric structure containing all of its diiron sites. Iron(II) apparently stabilizes the native or a nearly native structure during reconstitution. OxymyoHr and oxyHr are the major initial products of reconstitution. The yield of oxymyoHr from apomyoHr was approximately 87%. In contrast to reconstituted oxymyoHr, where essentially all of the iron appears to be functional, approximately 30% of the diiron sites in the reconstituted oxyHr are unable to bind O2 at ambient p(O2).(ABSTRACT TRUNCATED AT 250 WORDS)

We report the purification and characterization of a soluble cytochrome b5 from coelomic erythrocytes of the sipunculan worm, Phascolopsis gouldii. We also report the isolation and purification of a membrane-bound NADH-cytochrome-b5 reductase from these erythrocytes. The non-heme iron protein, hemerythrin (Hr), is known to be the oxygen carrier in these erythrocytes. The aforementioned purified cytochrome b5 and reductase together catalyze the reduction of P. gouldii [Fe(III),Fe(III)]metHr to [Fe(II),Fe(II)deoxyHr by NADH. EPR spectroscopy demonstrates that a redox process involving formation of the intermediate [Fe(II),Fe(III)]semi-metHr occurs within intact sipunculan erythrocytes as well as in the system of purified components. The rhombic g-tensor of the EPR signal in both cases resembles that of (semi-met)RHr, the form obtained by one-electron reduction of metHr. These observations suggest that cytochrome b5 and NADH-cytochrome-b5 reductase in sipunculan erythrocytes function to counteract autoxidation of oxyHr. The sequence of electron flow in the system of purified components is: NADH----NADH-cytochrome-b5 reductase----cytochrome b5----metHr. At pH 7.5, the reduction of metHr in this system occurs in two phases, only the first of which is dependent on concentration of cytochrome b5. From an analysis of the kinetics and the EPR time-course, we propose that the two phases represent sequential reduction of met- to semi-metHr and reduction of semi-metHr to deoxyHr. This report represents the first demonstration of a physiological system for reduction of metHr.

We have examined the effects on redox kinetics of changing the reduction potential of the mu-oxo-bridged binuclear iron center in octameric hemerythrin (Hr) from Phascolopsis gouldii. The opportunity to examine such effects is provided by the availability of mu-sulfidomethemerythrin (mu-S2-metHr), whose [Fe(III),Fe(III)]met----[Fe(II),Fe(III)]semi-met reduction potential is approximately 200 mV higher than that of methemerythrin (metHr). We have used, as redox partners to Hr, a set of metal complexes and the heme proteins deoxymyoglobin (Mb) and cytochrome b5. The latter protein from P. gouldii is a presumed physiological redox partner of Hr. Similar kinetics at pH 8 in the presence or absence of the allosteric effector perchlorate suggest reduction of the iron atom closer to the outer surface of each subunit in the Hr octamer during the met----semi-met transformation. For all reducing agents, the experimentally determined ratio of second-order rate constants for reductions of mu-S2-metHr and metHr, k12(mu-S2-met)/k12(met), is close to the value of 40 predicted by the simple Marcus relation for "outer-sphere" electron transfer. For oxidations of (semi-met)RHr and mu-S2-semi-metHr, the predicted value of 40 for k12[(semi-met)R]/k12(mu-S2-semi-met) is closely approximated when Fe(CN)6(3-) is used as oxidant. The ionic strength dependence of the second-order rate constant suggests electrostatic interactions of opposite charges during reduction of metHr by P. gouldii cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

Conclusive evidence is presented for an S = 1/2 spincoupled pair of high spin ferric and ferrous ions in the major reaction product of sulfide with the met form of the non-heme iron oxygen-carrying protein hemerythrin. Evidence for an analogous selenide derivative is also reported. Mössbauer and EPR spectroscopy establish (a) the charge and spin states of the individual iron atoms in sulfidehemerythrin as Fe(III), S = 5/2, and Fe(II), S = 2, and (b) the existence of an antiferromagnetic exchange interaction that couples the two spins to a resultant spin S = 1/2. The combined Mössbauer and EPR data confirm the correctness of the formulation first proposed for semi-methemerythrin by Harrington, P.C., de Waal, D.J.A., and Wilkins, R.G. ((1978) Arch. Biochem. Biophys. 191, 444-451) and furthermore show that a majority of the iron centers in the protein can be stabilized at this oxidation level. The results also demonstrate a new route to semi-methemerythrin. A titration of methemerythrin with selenide indicates that this derivative forms by a two step process consisting of first, reduction to the semi-met oxidation level by selenide and second, binding of selenide to either one or both irons.

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.

In anaerobic phosphate buffer, pH 6.3-7.5, deoxyhemerythrin is oxidized to semi-methemerythrin (semi-met) by excess sodium nitrite. This oxidation is quantitative as judged by EPR spectroscopy. Further oxidation to methemerythrin is not detected. The absorbance changes of hemerythrin during the oxidation are biphasic. The rate of the faster first phase is linearly dependent on [H+] and [NO2-] suggesting that the oxidant is nitrous acid rather than nitrite. During the slower second phase, the characteristic EPR spectrum of semi-methemerythrin appears. The first phase can be interpreted by a scheme in which nitrous acid transforms deoxyhemerythrin (FeIIFeII) to the semi-met nitrosyl adduct (FeIIFeIIINO) and hydroxide. Independent experiments confirm that the combination of semi-met plus NO produces an EPR-silent adduct. The rates of the absorbance changes for the second phase are nearly independent of nitrite concentration and pH in the range 6.3-7.5. This slower phase involves the transformation of the EPR-silent intermediate to the semi-met nitrite adduct (FeIIFeIIINO2-) and is consistent with rate-limiting dissociation of nitric oxide followed by rapid attachment of nitrite. Nitrite appears to be a unique oxidant of deoxyhemerythrin in that when employed in excess, the final, stable product is semi-met- rather than methemerythrin. The lack of reactivity of ethyl nitrite with deoxyhemerythrin suggests that HONO oxidizes deoxyhemerythrin via an "inner-sphere" process in contrast to oxidants such as Fe(CN)6(3-). A proposed generalization is that excesses of "inner-sphere" oxidants convert deoxy to (semi-met)R, which is stabilized with respect to (semi-met)R, which is stabilized with respect to (semi-met)0 and met because the oxidant and/or a product of the oxidant can bind to the iron site.

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.

Nitric oxide forms adducts with the binuclear iron site of hemerythrin (Hr) at [Fe(II),Fe(II)]deoxy and [Fe(II),Fe(III)]semimet oxidation levels. With deoxyHr our results establish that (i) NO binds reversibly, forming a complex which we label deoxyHrNO, (ii) NO forms a similar but distinct complex in the presence of fluoride, which we label deoxyHrFNO, (iii) NO is directly coordinated to one iron atom of the binuclear pair in these adducts, most likely in a bent end-on fashion, and (iv) the iron atoms in the binuclear sites of both deoxyHrNO and deoxyHrFNO are antiferromagnetically coupled, thereby generating unique electron paramagnetic resonance (EPR) detectable species. The novel EPR signal of deoxyHrNO (deoxyHrFNO) with g[[ = 2.77 (2.58) and g = 1.84 (1.80) is explained by the magnetic interaction of the Fe(II) (S' = 2) and [FeNO]7 (S = 3/2) centers observed by Mössbauer spectroscopy. Antiferromagnetic coupling leads to a ground state of Seff = 1/2. Analysis of the EPR parameters using the isotropic spin-exchange Hamiltonian, Hex = 2JS3/2.S2, and including zero-field splitting leads to a coupling constant, -J approximately 23 cm-1, for deoxyHrNO. The resonance Raman spectrum of deoxyHrNO shows features at 433 and 421 cm-1 that shift downward with 15N16O and that are assigned to stretching and bending modes, respectively, of the [FeNO]7 unit. Sensitivity of the bending mode to D2O suggests that bound NO participates in hydrogen bonding. We propose that the terminal oxygen atom of NO is hydrogen bonded to the proton of the mu-hydroxo bridge in the Fe-(OH)-Fe unit. A bent Fe-N-O geometry is supported by spectroscopic and structural comparisons to synthetic complexes and is consistent with a limiting [FeII,FeIIINO-] formulation for deoxyHrNO. Reversibility of NO binding to deoxyHr is demonstrated by bleaching of the optical and EPR spectra of deoxyHrNO upon additions of excess N3- or CNO-. DeoxyHrNO undergoes autoxidation under anaerobic conditions over the course of several hours. The product of this autoxidation appears to be an EPR-silent NO adduct of semimetHr. The formal one-electron oxidations of the binuclear iron site of deoxyHr by NO and by HNO2 can conceivably occur with no net change in charge on the iron site. In contrast, autoxidation of oxy- to metHr requires a change in net charge on the iron site, which may provide a kinetic barrier.