Unusual heme binding in the bacterial iron response regulator protein: spectral characterization of heme binding to the heme regulatory motif.
ABSTRACT: We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.
Project description: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-erb?. 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-erb? 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.
Project description:The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.
Project description:The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys(94) is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys(94) is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.
Project description: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 ?-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.
Project description:Rev-erb? is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes. Heme binding to Rev-erb? indirectly facilitates its interaction with the nuclear receptor co-repressor (NCoR1), resulting in repression of Rev-erb? target genes. Fe3+-heme binds in a 6-coordinate complex with axial His and Cys ligands, the latter provided by a heme-regulatory motif (HRM). Rev-erb? was thought to be a heme sensor based on a weak Kd value for the Rev-erb?·heme complex of 2 ?m determined with isothermal titration calorimetry. However, our group demonstrated with UV-visible difference titrations that the Kd value is in the low nanomolar range, and the Fe3+-heme off-rate is on the order of 10-6 s-1 making Rev-erb? ineffective as a sensor of Fe3+-heme. In this study, we dissected the kinetics of heme binding to Rev-erb? and provided a Kd for Fe3+-heme of ?0.1 nm Loss of the HRM axial thiolate via redox processes, including oxidation to a disulfide with a neighboring cysteine or dissociation upon reduction of Fe3+- to Fe2+-heme, decreased binding affinity by >20-fold. Furthermore, as measured in a co-immunoprecipitation assay, substitution of the His or Cys heme ligands in Rev-erb? was accompanied by a significant loss of NCoR1 binding. These results demonstrate the importance of the Rev-erb? HRM in regulating interactions with heme and NCoR1 and advance our understanding of how signaling through HRMs affects the major cellular processes of circadian rhythm maintenance and metabolism.
Project description:Cystathionine ?-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ?(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.
Project description: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.
Project description: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 ?-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.
Project description:MauG is a diheme enzyme that oxidizes two protein-bound tryptophan residues to generate a catalytic tryptophan tryptophylquinone cofactor within methylamine dehydrogenase. Upon the two-electron oxidation of bis-ferric MauG, the two c-type hemes exist as a spin-uncoupled bis-Fe(IV) species with only one binding oxygen, which is chemically equivalent to a single ferryl heme plus a pi porphyrin cation radical ( Li , X. et al. ( 2008 ) Proc. Natl. Acad. Sci. U.S.A. 105 , 8597 - 8600 ). The EPR spectrum of the nitrosyl complex of fully reduced MauG shows a single six-coordinate Fe(II)-NO species, which is characteristic of a histidine-ligated Fe(II)-NO moiety in the heme environment. Exposure of partially reduced MauG to NO reveals a redox equilibrium with facile electron transfer between hemes but with only one binding nitric oxide. Thus, the second heme is able to stabilize all three redox states of iron (Fe(II), Fe(III), and Fe(IV)) in a six-coordinate protein-bound heme without binding exogenous ligands. This is unprecedented behavior for a protein-bound heme for which each of these redox states is relevant to the overall catalytic mechanism. The results also illustrate the electronic communication between the two iron centers, which function as a diheme unit rather than independent heme cofactors.
Project description:It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450 ? P420 transition. Here, we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm?¹, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm?¹ is also inconsistent with a generalization of the suggestion that the 221 cm?¹ Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm?¹ mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine-bound heme systems. Additionally, the inverse correlation plots of the ?Fe-His and ?CO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO-bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of an HXC-Fe motif in many CYP systems allows the C ? H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix, and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found), a somewhat larger conformational change would be necessary to recuit a nearby histidine.