Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase.

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex in vitro. We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.

Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells.

Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling.

Regulation of protein function and degradation by heme, heme responsive motifs, and CO.

Heme is an essential biomolecule and cofactor involved in a myriad of biological processes. In this review, we focus on how heme binding to heme regulatory motifs (HRMs), catalytic sites, and gas signaling molecules as well as how changes in the heme redox state regulate protein structure, function, and degradation. We also relate these heme-dependent changes to the affected metabolic processes. We center our discussion on two HRM-containing proteins: human heme oxygenase-2, a protein that binds and degrades heme (releasing Fe2+ and CO) in its catalytic core and binds Fe3+-heme at HRMs located within an unstructured region of the enzyme, and the transcriptional regulator Rev-erbß, a protein that binds Fe3+-heme at an HRM and is involved in CO sensing. We will discuss these and other proteins as they relate to cellular heme composition, homeostasis, and trafficking. In addition, we will discuss the HRM-containing family of proteins and how the stability and activity of these proteins are regulated in a dependent manner through the HRMs. Then, after reviewing CO-mediated protein regulation of heme proteins, we turn our attention to the involvement of heme, HRMs, and CO in circadian rhythms. In sum, we stress the importance of understanding the various roles of heme and the distribution of the different heme pools as they relate to the heme redox state, CO, and heme binding affinities.

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle-when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.

Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site. Outside their catalytic cores are two regions unique to HO2: a 20-amino acid-long N-terminal extension and a C-terminal domain containing two heme regulatory motifs (HRMs) that bind heme independently of the core. Both HO isoforms contain a C-terminal hydrophobic membrane anchor; however, their sequences diverge. Here, using hydrogen-deuterium exchange MS, size-exclusion chromatography, and sedimentation velocity, we investigated how these divergent regions impact the dynamics and structure of the apo and heme-bound forms of HO1 and HO2. Our results reveal that heme binding to the catalytic cores of HO1 and HO2 causes similar dynamic and structural changes in regions (proximal, distal, and A6 helices) within and linked to the heme pocket. We observed that full-length HO2 is more dynamic than truncated forms lacking the membrane-anchoring region, despite sharing the same steady-state activity and heme-binding properties. In contrast, the membrane anchor of HO1 did not influence its dynamics. Furthermore, although residues within the HRM domain facilitated HO2 dimerization, neither the HRM region nor the N-terminal extension appeared to affect HO2 dynamics. In summary, our results highlight significant dynamic and structural differences between HO2 and HO1 and indicate that their dissimilar C-terminal regions play a major role in controlling the structural dynamics of these two proteins.

An unlikely heme chaperone confirmed at last.

Labile heme, as opposed to heme that is tightly bound within proteins, is thought to require a chaperone to be trafficked within the cell due to its cytotoxicity, but the identity of this chaperone was not known. A new study reveals that an unlikely protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is a heme chaperone that binds and transfers labile heme to downstream target proteins. These results provide a new framework for understanding heme homeostasis and raise intriguing questions regarding the intersection of heme transport, carbohydrate metabolism, and intracellular signaling.

Redox Regulation of Heme Oxygenase-2 and the Transcription Factor, Rev-Erb, Through Heme Regulatory Motifs.

SIGNIFICANCE: Heme binds to and serves as a cofactor for a myriad of proteins that are involved in diverse biological processes. Hemoproteins also exhibit varying modes of heme binding, suggesting that the protein environment contributes to the functional versatility of this prosthetic group. The subject of this review is a subset of hemoproteins that contain at least one heme regulatory motif (HRM), which is a short sequence containing a Cys-Pro core that, in many cases, binds heme with the Cys acting as an axial ligand. Recent Advances: As more details about HRM-containing proteins are uncovered, some underlying commonalities are emerging, including a role in regulating protein stability. Further, the cysteines of some HRMs have been shown to form disulfide bonds. Because the cysteines must be in the reduced, dithiol form to act as a heme axial ligand, heme binds at these sites in a redox-regulated manner, as demonstrated for heme oxygenase-2 (HO2) and Rev-erbß. CRITICAL ISSUES: HRM-containing proteins have wide variations in heme affinity, utilize different axial ligand schemes, and exhibit differences in the ability to act as a redox sensor-all while having a wide variety of biological functions. Here, we highlight HO2 and Rev-erbß to illustrate the similarities and differences between two hemoproteins that contain HRMs acting as redox sensors. FUTURE DIRECTIONS: HRMs acting as redox sensors may be applicable to other HRM-containing proteins as many contain multiple HRMs and/or other cysteine residues, which may become more evident as the functional significance of HRMs is probed in additional proteins.

Comparison of the Mechanisms of Heme Hydroxylation by Heme Oxygenases-1 and -2: Kinetic and Cryoreduction Studies.

The two isoforms of human heme oxygenase (HO1 and HO2) catalyze oxidative degradation of heme to biliverdin, Fe, and CO. Unlike HO1, HO2 contains two C-terminal heme regulatory motifs (HRMs) centered at Cys265 and Cys282 that act as redox switches and, in their reduced dithiolate state, bind heme (Fleischhacker et al., Biochemistry , 2015 , 54 , 2693 - 2708 ). Here, we describe cryoreduction/annealing and electron paramagnetic resonance spectroscopic experiments to study the structural features of the oxyheme moiety in HO2 and to elucidate the initial steps in heme degradation. We conclude that the same mechanism of heme hydroxylation to α-meso-hydroxyheme is employed by both isoforms and that the HRMs do not affect the physicochemical properties of the oxy-Fe(II) and HOO-Fe(III) states of HO2. However, the absorption spectrum of oxy-Fe(II)-HO2 is slightly blue-shifted relative to that of HO1. Furthermore, heme hydroxylation proceeds three times more slowly, and the oxy-Fe(II) state is 100-fold less stable in HO2 than in HO1. These distinctions are attributed to slight structural variances in the two proteins, including differences in equilibrium between open versus closed conformations. Kinetic studies revealed that heme oxygenation by HO2 occurs solely at the catalytic core in that a variant of HO2 lacking the C-terminal HRM domain exhibits the same specific activity as one containing both the catalytic core and HRM domain; furthermore, a truncated variant containing only the HRM region binds but cannot oxidize heme. In summary, HO1 and HO2 share similar catalytic mechanisms, and the HRMs do not play a direct role in the HO2 catalytic cycle.

The C-terminal heme regulatory motifs of heme oxygenase-2 are redox-regulated heme binding sites.

Heme oxygenase-2 (HO2), an enzyme that catalyzes the conversion of heme to biliverdin, contains three heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1-288 (HO2sol) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2sol via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe(3+)-heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of heme to disulfide-reduced HO2sol, in the work described here, we further investigated the properties of Fe(3+)-heme bound to HO2. Specifically, we investigated binding of Fe(3+)-heme to a truncated form of soluble HO2 (residues 213-288; HO2tail) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2tail in the disulfide-reduced state binds Fe(3+)-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2sol that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2sol binds one Fe(3+)-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2sol binds slightly more than two. Both Cys265 and Cys282 were identified as Fe(3+)-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe(3+)-heme binds with a much weaker affinity to Cys282 than to Cys265, which has an affinity much weaker than that of the His45 binding site in the catalytic core. In summary, disulfide-reduced HO2 has multiple binding sites with varying affinities for Fe(3+)-heme.

Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme.

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O2- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2(O)), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2(R)) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme.

Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli.

Fe-S clusters are essential across the biological world, yet how cells regulate expression of Fe-S cluster biogenesis pathways to cope with changes in Fe-S cluster demand is not well understood. Here, we describe the mechanism by which IscR, a [2Fe-2S] cluster-containing regulator of Escherichia coli, adjusts the synthesis of the Isc Fe-S biogenesis pathway to maintain Fe-S homeostasis. Our data indicate that a negative feedback loop operates to repress transcription of the iscRSUA-hscBA-fdx operon, encoding IscR and the Isc machinery, through binding of [2Fe-2S]-IscR to two upstream binding sites. IscR was shown to require primarily the Isc pathway for synthesis of its Fe-S cluster, providing a link between IscR activity and demands for Fe-S clusters through the levels of the Isc system. Surprisingly, the isc operon was more repressed under anaerobic conditions, indicating increased Fe-S cluster occupancy of IscR and decreased Fe-S cluster biogenesis demand relative to aerobic conditions. Consistent with this notion, overexpression of a Fe-S protein under aerobic conditions, but not under anaerobic conditions, led to derepression of P(iscR). Together, these data show how transcriptional control of iscRSUA-hscBA-fdx by [2Fe-2S]-IscR allows E. coli to respond efficiently to varying Fe-S demands.

Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR.

IscR is an Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mössbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S](+) cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the [2Fe-2S](2+) form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and nuclear magnetic resonance spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved His107 residue is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.

Does activation of the anti proton, rather than concertedness, determine the stereochemistry of base-catalyzed 1,2-elimination reactions? Anti stereospecificity in E1cB eliminations of ß-3-trifluoromethylphenoxy esters, thioesters, and ketones.

As part of a comprehensive investigation on the stereochemical aspects of base-catalyzed 1,2-elimination reactions, we have studied a set of acyclic carbonyl substrates that react by an irreversible E1cB mechanism with largely anti stereospecificity. (2)H NMR data show that these reactions using KOH in EtOH/H(2)O under non-ion-pairing conditions produce a minimum of 85-89% anti elimination on stereospecifically labeled tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanoate, S-tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanethioate, and the related ketones, (4R*,5R*)- and (4R*,5S*)-5-(3-trifluoromethylphenoxy)-4,5-(2)H(2)-3-hexanone. With both diastereomers of each substrate available, the KIEs can be calculated and the innate stereoselectivities determined. The elimination reactions of the ß-3-trifluoromethylphenoxy substrates occur by E1cB mechanisms with diffusionally equilibrated enolate-anion intermediates. Thus, it is clear that anti elimination does not depend solely upon concerted E2 mechanisms. Negative hyperconjugation provides a satisfactory explanation for the anti stereospecificity exhibited by our carbonyl substrates, where the leaving group activates the anti proton, leading to the enolate intermediate. The activation of the anti proton by negative hyperconjugation may also play a role in the concerted pathways of E2 mechanisms. We have also measured the rates of the hydroxide-catalyzed elimination reactions of butanoate, thiobutanoate, and ketone substrates in EtOH/H(2)O, with ß-tosyloxy, acetoxy, and 3-trifluoromethylphenoxy nucleofuges.

Iron-containing transcription factors and their roles as sensors.

Iron-binding transcription factors are widespread throughout the bacterial world and to date are known to bind several types of cofactors, such as Fe2+, heme, or iron-sulfur clusters. The known chemistry of these cofactors is exploited by transcription factors, including Fur, FNR, and NsrR, to sense molecules such as Fe2+, gases (e.g. oxygen and nitric oxide), or reactive oxygen species. New structural data and information generated by genome-wide analysis studies have provided additional details about the mechanism and function of iron-binding transcription factors that act as sensors.

Spectroscopic and computational characterization of the base-off forms of cob(II)alamin.

The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center. Accurate spin Hamiltonian parameters for these low-spin Co(2+) centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered pi --> pi* and Co-based d --> d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co(2+)Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co(2+)Cbl species, the unpaired spin density is primarily localized in the Co 3d(z(2))-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin-orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co(2+)Cbl --> Co(1+)Cbl reduction.

Ligand trans influence governs conformation in cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme. Reductive methylation with flavodoxin and adenosylmethionine returns the enzyme to an active methylcobalamin state. At different points during the reaction cycle, the coordination state of the cobalt of the cobalamin changes. The imidazole side chain of His759 coordinates to cobalamin in a "His-on" state and dissociates to produce a "His-off" state. The His-off state has been associated with a conformation of MetH that is poised for reactivation of cobalamin by reductive methylation rather than catalysis. Our studies on cob(III)alamins bound to MetH, specifically aqua-, methyl-, and n-propylcobalamin, show a correlation between the accessibility of the reactivation conformation and the order of the established ligand trans influence. The trans influence also controls the affinity of MetH in the cob(III)alamin form for flavodoxin. Flavodoxin, which acts to shift the conformational equilibrium toward the reactivation conformation, binds less tightly to MetH when the cob(III)alamin has a strong trans ligand and therefore has less positive charge on cobalt. These results are compared to those for cob(II)alamin MetH, illustrating that access to the reactivation conformation is governed by the net charge on the cobalt as well as the trans influence in cob(III)alamins.

Probing the role of the histidine 759 ligand in cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH.

Conversion of aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase, a metalloenzyme, into a nonmetalloenzyme.

The Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a class II metalloenzyme, is converted into an active nonmetalloenzyme by a single amino acid mutation, namely, C11N. The result may provide insight into the evolutionary link between the two KDO8PS classes as well as the potential role of the metal and/or asparagine in the catalytic mechanism.