In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the 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 to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge.
View Article and Find Full Text PDFAldoxime 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.
View Article and Find Full Text PDFHemes (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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFSeveral 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.
View Article and Find Full Text PDFThe heme-based aerotactic transducer (HemAT) is an oxygen-sensor protein consisting of a sensor and a signaling domain in the N- and C-terminal regions, respectively. Time-resolved step-scan FTIR spectroscopy was employed to characterize protein intermediate states obtained by photolysis of the carbon monoxide complexes of sensor-domain, full-length HemAT, and the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) HemAT mutants. We assign the spectral components to discrete substructures, which originate from a helical structure that is solvated (1638 cm) and a native helix that is protected from solvation by interhelix tertiary interactions (1654 cm).
View Article and Find Full Text PDFHemAT 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.
View Article and Find Full Text PDFIron(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.
View Article and Find Full Text PDFCooA 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.
View Article and Find Full Text PDFCorynebacteria 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.
View Article and Find Full Text PDFMalaria 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.
View Article and Find Full Text PDFSensor 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.
View Article and Find Full Text PDFBiochem Biophys Res Commun
October 2013
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.
View Article and Find Full Text PDFRr-CooA is a CO-sensor heme protein, where binding of CO with the heme group stimulates a transcriptional activator activity of CooA. In this process, the heme undergoes a series of ligand exchanges. In the ferric form, the heme has Cys75 and Pro2 as the axial ligands.
View Article and Find Full Text PDFAlthough heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear.
View Article and Find Full Text PDFThe crystal structure of a truncated Aer2, a signal transducer protein from Pseudomonas aeruginosa, consisting of the heme-containing PAS and di-HAMP domains revealed that a distal tryptophan residue (Trp283) plays an important role in stabilizing the heme-bound O(2) and intra-molecular signal transduction upon O(2) binding.
View Article and Find Full Text PDFHemAT 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.
View Article and Find Full Text PDFHemAT-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.
View Article and Find Full Text PDFFourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.
View Article and Find Full Text PDFAntioxid Redox Signal
April 2012
Significance: Gas molecules function as signaling molecules in many biological regulatory systems responsible for transcription, chemotaxis, and other complex physiological processes. Gas sensor proteins play a crucial role in regulating such biological systems in response to gas molecules.
Recent Advances: New sensor proteins that sense oxygen or nitric oxide have recently been found, and they have been characterized by X-ray crystallographic and/or spectroscopic analysis.
Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (μ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.
View Article and Find Full Text PDFTranscriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii. In the present study, we have purified full-length VnfA produced in E. coli as recombinant proteins (Strep-tag attached and tag-less proteins), enabling detailed characterization of VnfA for the first time.
View Article and Find Full Text PDFWe 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.
View Article and Find Full Text PDFAldoxime 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.
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