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In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference for defining a threshold used for grouping the isomorphous datasets (‘isomorphic threshold’) poses a challenge. Here, we have applied unit cell-based and intensity-based HCAs to the datasets of apo-trypsin and inhibitor-bound trypsin that were mixed post-data acquisition to investigate how effective HCA is in classifying polymorphous datasets. Single-step intensity-based HCA successfully classified polymorphs with a certain ‘isomorphic threshold’. In datasets of several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested ‘isomorphic threshold’. Polymorphs were also detected in single crystals using the data collected by the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.

We have determined the crystal structure of thermoglobin (AaTgb) from a hyperthermophilic bacterium Aquifex aeolicus. Tyrosine and glutamine at the B10 and E7 position, respectively, are conserved ...

Since the bullfrog H-ferritin L134P mutant in which leucine 134 is replaced with proline was found to exhibit a flexible conformation in the C3 axis channel, homologous ferritins with the corresponding mutation have often been studied in terms of a mechanism of iron release from the mineral core within the protein cavity. Meanwhile, a ferritin mutant with the flexible channel is an attractive material in developing a method to encapsulate functional molecules larger than mononuclear ions into the protein cavity. This study describes the clathrate with a horse spleen L-ferritin L134P mutant containing Prussian blue (PB) without a frequently used technique, disassembly and reassembly of the protein subunits. The spherical shell of ferritin was confirmed in a TEM image of the clathrate. The produced clathrate (PB@L134P) was soluble in water and reproduced the spectroscopic and electrochemical properties of PB prepared using the conventional method. The catalytic activity for an oxidoreductive reaction with H2O2, one of the major applications of conventional PB, was also observed for the clathrate. The instability of PB in alkaline solutions, limiting its wide applications in aqueous media, was significantly improved in PB@L134P, showing the protective effect of the protein shell. The method developed here shows that horse spleen L-ferritin L134P is a useful scaffold to produce clathrates of three-dimensional complexes with ferritin.

Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size. Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN− are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N10-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis., Muraki et al. determine the crystal structures of HypX, which is an accessory protein required to assemble the active site of NiFe hydrogenase, to investigate the mechanism of carbon monoxide (CO) biosynthesis. This study suggests the reaction scheme of CO biosynthesis and provides insight into how CoA is accommodated during this process.

Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction., Nishinaga et al. present structural characterization of the transcription regulator PefR from S. agalactiae in different states (apo-, DNAbound, heme-bound, CO-heme-bound and CN-heme-bound-PefRs). Structural comparison revealed that unique heme coordination controls structural rearrangement for the survival of the neonatal infection-causing hemolytic bacteria.

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the ν(Fe-His) mode, arising from the proximal His77-iron stretch, does not shift until 50 μs after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 μs after the photodissociation of CO, suggesting that the ν(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon CO binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 μs after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.

The heme-based sensor proteins become a new family of hemeproteins that show a new biological function as the sensor for gas molecules such as O2, CO, and NO.These gas molecules can act as signaling molecules for the regulation of biological signal transduction systems. These regulatory systems consist of a sensor and regulatory domains/proteins, in which the sensor domain/protein senses the physiological gas molecule and then regulates the biological function of the regulatory domain/protein via intra- or inter-molecular signal transduction between the sensor and regulatory domains/proteins. The heme-based sensor domain/protein senses a gas molecule as its physiological effector by binding it to the heme in the sensor domain/protein. Though the heme-based sensor proteins adopt several different domains including H-NOX, globin, PAS, and GAF domains as the sensor domain in which the heme is accommodated, it is common among the heme-based sensors/proteins that gas-binding to the heme triggers the signal transduction process to regulate the regulatory domain/protein, which is initiated by a conformational change around the heme upon gas-binding. The interactions between the heme-bound ligand and surrounding amino acid residues will play an important role for distinguishing the physiological gas molecule among other heme-binding ligands and for the conformational change responsible for signal transduction. Thus, structural information of the sensor domain/protein is essential to elucidate the molecular mechanisms of gas-sensing and functional regulation by the heme-based sensor proteins. In this work, we have elucidated the structure-function relationships of several heme-based sensor proteins including HemAT, Aer2, HemDGC. While HemAT and Aer2 are signal transducer proteins in the aerotaxis regulatory systems, HemDGC is a heme-containing diguanylate cyclase that consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. We have determined the X-ray crystal structures of Aer2-N384 (residues 1-384 that consists of three-HAMP, PAS, and di-HAMP) and Aer2-PH (residues 173-384 that consists of PAS and di-HAMP) to elucidate the mechanism by which intramolecular signal transduction proceeds between the HAMP and PAS domains in Aer2. Aer2-N384 is a homodimer having a non-crystallographic 2-fold symmetry. The three-unit poly-HAMP, PAS, and di-HAMP domains are ordered in linear configuration. A heme exists in a hydrophobic pocket in the PAS domain. His234 serves as the proximal ligand of the heme. The structural analyses of Aer2 suggests that the heme-bound O2 forms a hydrogen bond with Trp283. Trp283 is located on the C-terminal of the strand b5. This strand connects to the helix a5, which is the starting region of the C-terminal di-HAMP domain, suggesting that the hydrogen bond between Trp283 and O2 are responsible for intramolecular signal transduction. The enzymatic activity of HemDGC is regulated by the ligand binding to the heme. The enzymatic activity for the formation of c-di-GMP from GTP was measured for ferric, cyanomet, deoxy, oxy, CO-bound, and NO-bound HemDGC. Only oxy HemDGC showed the activity of diguanylate cyclase for the formation of c-di-GMP from GTP, but ferric, cyanomet, deoxy, CO-bound, and NO-bound HemDGC did not at all. These results indicate that O2 will be a physiological effector of HemDGC and will regulate the enzymatic activity of HemDGC via the formation of the oxy-complex of the heme. Thus, the globin domain of HemDGC acts as a sensor that can strictly discriminate O2 among external ligands capable to bind to the heme. HemDGC shows different hydrogen bonding patterns between the O2- and CO-bound forms. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O2, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O2 sensing responsible for the regulation of the enzymatic activity.

Iron(iii) carbonyl complexes are stabilized by a pincer ligand containing pyridine-N, phenyl-C and thiolate-S donors and two axial phosphine ligands. The N,C,S-pincer iron(iii) carbonyl complexes show CO-releasing properties induced by visible light.

This mini-review presents a recent development of a new function of heme as a signaling molecule especially in the regulation of gene expression. Heme is biosynthesized as a prosthetic group for heme proteins, which play crucial roles for respiration, photosynthesis, and many other metabolic reactions. In some bacteria, exogenous heme molecules are used as a heme or an iron sources to be uptaken into cytoplasm. As free heme molecules are cytotoxic, the intracellular concentrations of biosynthesized or uptaken heme should be strictly controlled. In this mini-review, we summarize the biochemical and biophysical properties of the transcriptional regulators and heme-sensor proteins responsible for these regulatory systems to maintain heme homeostasis.

Though CO is well known as a respiratory poison, it becomes apparent that it has positive biological functions in various systems. CO acts as a component of the active sites in hydrogenase metalloenzymes, but also as a signalling molecule in bacteria and mammals. In this chapter, endogenous CO production systems and biological utilization of CO are described briefly and then followed by discussion of the bacterial and mammalian sensor proteins that are required for CO to act as a signalling molecule. These proteins are responsible for the regulation of transcription or enzymatic activity in response to CO. All CO-sensor proteins identified to date utilize the haem group to sense CO. As haem is a good spectroscopic probe, detailed characterizations have utilized spectroscopic techniques along with X-ray crystallography. The structural and functional relationships of CO-sensor proteins CooA, RcoM, NPAS2, CLOCK, and CBS, are discussed.

Typical signal-transduction systems and prosthetic groups for gas sensing are summarized. Biological signal-transduction systems can be classified into single-, two- and multicomponent systems, based on the number(s) of proteins constituting the systems. Gas-sensor proteins are known in all three systems. While NO-, O2-, and CO-sensing transcriptional regulators and enzymes are known as members of the single-component systems, NO- and O2-sensing histidine kinases and cognate response regulators constitute the two-component systems. Bacterial chemotaxis regulatory systems, mammalian O2 sensing systems mediated by the hypoxia inducible factors, and plant ethylene signalling systems are multicomponent systems discussed in this book. Haem, iron–sulfur clusters and mononuclear or binuclear nonhaem iron centres are typical metal-containing prosthetic groups employed as active sites for sensing gas molecules. Their basic properties are also summarized in this chapter.

HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.

The HupDCG protein complex is a putative ABC-transporter for heme in the pathogenic Gram-positive bacterium Listeria monocytogenes, where HupD functions as a heme-binding protein. UV–vis absorption...

Mammalian 5-aminolevulinic acid synthase 1 (ALAS1), an isozyme expressed in all cell types, catalyzes the first reaction in the heme biosynthetic pathway in mitochondria. Heme regulates ALAS1 funct...

Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O(2) sensor protein that acts as a chemotactic signal transducer. Binding of O(2) to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O(2)-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O(2) and CO dissociation. A comparison between the O(2) and CO complexes provides insights on O(2)/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10ns and 100μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe-His), for the O(2)-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O(2) dissociation would be associated with the O(2)/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O(2) dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O(2) dissociation are coupled with signal transduction in HemAT-Bs.

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced − CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O(2), but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O(2) sensing responsible for the regulation of the enzymatic activity.

Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R-CH=N-OH) to their corresponding nitrile (R-C triple bond N). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 A resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 A resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe(3+) heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O2-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr70 in the heme distal side and Tyr133 and Trp132 from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2, but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme-bound O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2 binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.

HemAT is a signal transducer protein responsible for aerotaxis control of some bacteria and archaea, which contains a heme-containing globin domain as the sensor of its physiological effector, O 2. The interaction between the heme-bound ligand and the surrounding amino acid residue(s) plays a crucial role for selective sensing of O 2 and signal transduction by HemAT. In this work, we have elucidated by resonance Raman spectroscopy how O 2 and CO interact with HemAT- Hs and HemAT- Rr , HemAT from Halobacterium salinarum and Rhodospirillum rubrum, respectively. HemAT- Hs and HemAT- Rr showed three conformers in the O 2-bound form, as is the case of HemAT- Bs , HemAT from Bacillus subtilis. Though the hydrogen bonding patterns observed in the three conformers were the same for HemAT- Bs , HemAT- Hs , and HemAT- Rr , the involved residues for the hydrogen bonding interaction were different from one another.

CooA is a CO-dependent transcriptional activator and transmits a CO-sensing signal to a DNA promoter that controls the expression of the genes responsible for CO metabolism. CooA contains a b-type heme as the active site for sensing CO. CO binding to the heme induces a conformational change that switches CooA from an inactive to an active DNA-binding form. Here, we report the crystal structure of an imidazole-bound form of CooA from Carboxydothermus hydrogenoformans (Ch-CooA). In the resting form, Ch-CooA has a six-coordinate ferrous heme with two endogenous axial ligands, the alpha-amino group of the N-terminal amino acid and a histidine residue. The N-terminal amino group of CooA that is coordinated to the heme iron is replaced by CO. This substitution presumably triggers a structural change leading to the active form. The crystal structure of Ch-CooA reveals that imidazole binds to the heme, which replaces the N terminus, as does CO. The dissociated N terminus is positioned approximately 16 A from the heme iron in the imidazole-bound form. In addition, the heme plane is rotated by 30 degrees about the normal of the porphyrin ring compared to that found in the inactive form of Rhodospirillum rubrum CooA. Even though the ligand exchange, imidazole-bound Ch-CooA remains in the inactive form for DNA binding. These results indicate that the release of the N terminus resulting from imidazole binding is not sufficient to activate CooA. The structure provides new insights into the structural changes required to achieve activation.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O2, from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O2-bound form, in addition to that between Thr95 and the heme-bound O2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O2. This stepwise conformational change would be essential to selective O2 sensing and signal transduction by HemAT-Bs.

Chemotaxis signal transducer protein DcrA from a sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was previously shown to contain a c-type heme in its periplasmic domain (DcrA-N) for sensing redox and/or oxygen [Fu et al. (1994) J. Bacteriol. 176, 344-350], which is the first example of a heme-based sensor protein containing a c-type heme as a prosthetic group. Optical absorption and resonance Raman spectroscopies indicates that heme c in DcrA-N shows a redox-dependent ligand exchange. Upon reduction, a water molecule that may be the sixth ligand of the ferric heme c is replaced by an endogenous amino acid. Although the reduced heme in DcrA-N is six-coordinated with two endogenous axial ligands, CO can easily bind to the reduced heme to form CO-bound DcrA-N. Reaction of the reduced DcrA-N with molecular oxygen results in autoxidation to form a ferric state without forming any stable oxygen-bound form probably due to the extremely low redox potential of DcrA-N (-250 mV). Our study supports the initial idea by Fu et al. that DcrA would act as a redox and/or oxygen sensor, in which the ligand exchange between water and an endogenous amino acid is a trigger for signal transduction. While the affinity of CO to DcrA-N (Kd = 138 microM) is significantly weak compared to those of other heme proteins, we suggest that CO might be another physiological effector molecule.

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.

CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration ( R g ) and the second virial coefficient ( A 2 ) were 25.3(±0.5) A and −0.39(±0.25)×10 −4 ml mol g −2 , respectively. CO binding caused a slight increase in R g (by 0.5 A) and a marked decrease in A 2 (by 5.09×10 −4 ml mol g −2 ). The observed decrease in A 2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of R g rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental R g and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8° around the axis of 2-fold symmetry.

Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E' of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the nu(Fe-CO) and nu(C-O) values in the resonance Raman and infrared spectra of the ferrous-CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The nu(Fe-O2) in the resonance Raman spectra of oxy Cgb can be observed at 572 cm(-1), suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.

The CO-sensing transcriptional activator CooA contains a protoheme that acts as a CO sensor. Mutagenesis and spectroscopic studies revealed the unique coordination structure of the heme in CooA, the Pro2/Cys75, Pro2/His77, and CO/His77 coordinations for the ferric, ferrous, and CO-bound hemes, respectively. The ligand exchange between Cys75 and His77 took place during the change in the oxidation state of the heme, which caused the hysteresis in redox titration of CooA. Furthermore, CO replaced Pro2 to activate CooA under physiological conditions. A truncated mutant, CooAΔN5, in which the first five residues in the N-terminal were deleted, showed similar spectroscopic and functional properties to those of the wild-type, indicating the flexibility of the polypeptide chain of CooA in its N-terminal region. In flash photolysis of CO-bound CooA, a biphasic geminate recombination process with the time constants of 78 and 386 ps was observed. The yield of the geminate recombination of CO was 90% for the CO-bound CooA.

Heme-based sensor proteins have become known to be a new member of hemeproteins in which the heme acts as the active center for sensing gas molecules such as O2, CO, and NO. HemAT and CooA are the O2 and CO sensor proteins, respectively. HemAT is a signal transducer protein in the aerotaxis control system in Bacillus subtilis. CooA is a transcriptional activator that regulates the expression of some proteins responsible for CO metabolism in Rhodospirillum rubrum. Here, the structure and function of HemAT and CooA are reviewed.

Heme uptake machinery of Corynebacteria including Corynebacteria glutamicum and Corynebacterium diphtheriae consists of heme binding proteins, HtaA and HtaB, and the ABC-type heme transporter HmuTUV. In this work, we have studied the structural and functional relationships of HtaA, HtaB and HmuT in Corynebacterium glutamicum. Sequence analysis identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB [1]. HtaA consists of two homologous CRs in the N- and C-terminal regions. We have determined the crystal structures of the N-, and C-terminal CR of HtaA (HtaA-N and HtaA-C, respectively) and HtaB at the resolution of 2.0, 1.3, and 1.7 Å, respectively. HtaA-N consists of 11 β strands and two short α helices and accommodates one heme molecule with Tyr58 located in the first α helix as the heme axial ligand. Tyr58 forms a hydrogen bond with His111. A heme propionate forms hydrogen bonds with Ser54 and Tyr201. Heme is accommodated in an open pocket formed by hydrophobic amino acid residues. HtaA-C and HtaB show similar global structures to HtaA-N. The key residues for heme-binding and recognition including the axial ligand of heme and residues involved in the hydrogen bonding interactions with heme are conserved among HtaA-N, HtaA-C, and HtaB. We also determined the crystal structure of HmuT at the resolution of 1.4 Å [2]. HmuT consists of structurally similar two domains located in the N-terminal and C-terminal regions connected a long α helix. A single heme molecule is bound in the cleft between these domains. Heme iron is ligated by His141 and Tyr240, and Tyr240 forms a hydrogen bond with Arg242. Intriguingly, HmuT binds a heme with two different orientations. References [1] C. E. Allen, M. P. Schmitt. (2011) J. Bacteriol. 193, 5374-5385. [2] N. Muraki, S. Aono. (2016) Chem. Lett. 45, 24-26.

Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum.

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.

CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.

The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2679-2687]. Although our previous resonance Raman study failed to detect the Fe-His stretching [nu(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to nu(Fe-His) at 211 cm(-)(1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low nu(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-)(1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.

FNR and SoxR are transcriptional regulators containing an iron–sulfur cluster. The iron–sulfur cluster in FNR acts as an oxygen sensor by reacting with oxygen. The structural change of the iron–sulfur cluster takes place when FNR senses oxygen, which regulates the transcriptional regulator activity of FNR through the change of the quaternary structure. SoxR contains the [2Fe–2S] cluster that regulates the transcriptional activator activity of SoxR. Only the oxidized SoxR containing the [2Fe–2S]2+cluster is active as the transcriptional activator. CooA is a transcriptional activator containing a protoheme that acts as a CO sensor. CO is a physiological effector of CooA and regulates the transcriptional activator activity of CooA. In this review, the biochemical and biophysical properties of FNR, SoxR, and CooA are described.

CooA is a transcriptional activator containing a b-type heme as a prosthetic group. The heme in CooA is in the six-coordinate form in the ferric and ferrous state. Although the ferrous heme is coordinated by two axial ligands, it can react easily with CO to form the CO-bound heme under physiological conditions. Replacement of the heme’s axial ligand by CO triggers the activation of CooA, which causes some conformational change around the heme and finally in the whole molecule. In this paper, the coordination structure of the heme in CooA and the mechanism of activation of CooA by CO were studied by spectroscopic analysis of the wild-type and some mutant CooAs. The amino acid residues responsible for the specific binding of CooA to target DNA were elucidated by site-directed mutagenesis.

The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO sensor in vivo. CooA is the first example of a transcriptional regulator containing a heme as a prosthetic group and of a hemeprotein in which CO plays a physiological role. In this study, we constructed an in vivo reporter system to measure the transcriptional activator activity of CooA and prepared some CooA mutants in which a mutation was introduced at Cys, His, Met, Lys, or Tyr. Only the mutations of Cys75 and His77 affected the electronic absorption spectra of the heme in CooA. The electronic absorption spectra, EPR spectra, and the transcriptional activator activity of the wild-type and mutant CooA proteins indicate that 1) the thiolate derived from Cys75 is the axial ligand in the ferric heme, but it is not coordinated to the CO-bound ferrous heme; 2) Cys75 is protonated or displaced in the ferrous heme; and 3) His77 is the proximal ligand in the CO-bound ferrous heme and probably also in the ferrous heme, but it is not coordinated to the ferric heme. NMR spectra reveal that the conformational change around the heme, which will trigger the activation of CooA by CO, takes place upon the binding of CO to the heme.

SoxR protein in Escherichia coli , which is a transcriptional activator for the transcription of soxS , contains four cysteine residues at positions 119, 122, 124 and 130. These cysteines have been separately mutated into serine by the site-directed mutagenesis. The wild type and the mutant SoxR proteins were expressed in E. coli JM109 with pKK223-3 based expression vectors containing a tac promoter system. Purified four cysteine-to-serine mutant SoxR proteins do not contain the iron–sulfur cluster though the wild type SoxR expressed in this system contains a [2Fe–2S] cluster, which shows that all of the four cysteine at positions 119, 122, 124 and 130 are the ligand of the [2Fe–2S] cluster in SoxR.

In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C=O stretching Raman lines of the CO-bound CooA were observed at 487 and 1969 cm−1, respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of ν(Fe-CO) implies an open conformation of the distal heme pocket, indicating that the ligand replaced by CO is located away from the bound CO. When the target DNA was added to CO-bound CooA, an appearance of a new ν(Fe-CO) line at 519 cm−1 and narrowing of the main line at 486 cm−1 were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.

Sensor proteins play crucial roles in maintaining homeostasis of cells by sensing changes in extra- and intracellular chemical and physical conditions to trigger biological responses. It has recently become clear that gas molecules function as signalling molecules in these biological regulatory systems responsible for transcription, chemotaxis, synthesis/hydrolysis of nucleotide second messengers, and other complex physiological processes. Haem-containing sensor proteins are widely used to sense gas molecules because haem can bind gas molecules reversibly. Ligand binding to the haem in the sensor proteins triggers conformational changes around the haem, which results in their functional regulation. Spectroscopic and crystallographic studies are essential to understand how these sensor proteins function in these biological regulatory systems. In this chapter, I discuss structural and functional relationships of haem-containing PAS and PAS-related families of the sensor proteins.

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.

The gene coding for a carbon monoxide-dependent transcriptional activator (cooA) in Rhodospirillum rubrum has been expressed in E. coli, and the recombinant CooA has been purified. CooA contains b-type heme which may act as a CO sensor in vivo. CO-bound CooA was formed when reduced CooA was reacted with CO, but not in the case of oxidized CooA. CooA is the first example of the heme protein acting as a DNA-binding transcriptional activator.