Cytoglobin (Cgb) is a recently discovered member of the vertebrate haem‐containing globin family. The structure of a new crystal form of wild‐type human Cgb (space group C2) was determined at a ...resolution of 1.68 Å. The results show the presence of an additional helix in the N‐terminal residues (4–20) prior to the A helix and an ordered loop structure in the C‐terminal region (168–188), while these extended peptides were invisible owing to disorder in the previously reported structures using a P3221 crystal at a resolution of 2.4 Å. A detailed comparison of the two crystal structures shows differences in the conformation of the residues (i.e. Arg84) in the haem environment owing to a different dimeric arrangement.
Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions ...have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.
Binding affinities for exogenous ligands were regulated by a disulfide bond in Cgb. The formation of the disulfide bond caused alteration of space of cavity, and the variation was also suggested by computational simulations. Remarkably, a structure with a disulfide bond showed 2.5 to 100-fold higher affinities for exogenous ligands. Display omitted
•Cytoglobin shapes monomers, dimers, and tetramers having distinct properties.•Monomeric cytoglobin forms two alternative structures with/without a disulfide bond.•Computational simulation could explain conformational changes by SS bond formation.•The physiological functions of cytoglobin may be regulated by the SS bond formation.
Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N2O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is ...produced from nitrite (NO2−) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.
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•An overexpression system for Pseudomonas aeruginosa cNOR was constructed.•Variants as well as wild-type cNOR can be purified using our expression system.•Facile in vivo screening for important residues in cNOR was established.•In vivo screening suggested a primary proton transfer pathway for cNOR catalysis.•Our expression system allows further functional studies of cNOR variants.
Fourier transform infrared (FTIR) spectra, “light” minus “dark” difference FTIR spectra, and time-resolved step-scan (TRS2) 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 H2O/D2O exchange and in the pH 5.6–8.5 range, suggesting the presence of two conformations at the active site. The observed C–O frequencies are 5 and 16 cm–1 lower and higher, respectively, than that obtained previously (Oinuma, K.-I.; et al. FEBS Lett. 2004, 568, 44–48). We suggest that the strength of the Fe–His bond and the neutralization of the negatively charged propionate groups modulate the ν(Fe–CO)/ν(CO) back-bonding correlation. The “light” minus “dark” difference FTIR spectra indicate that the heme propionates are in both the protonated and deprotonated forms, and the photolyzed CO becomes trapped within a ligand docking site (ν(CO) = 2138 cm–1). The TRS2-FTIR spectra show that the rate of recombination of CO to the heme is k 1945 cm–1 = 126 ± 20 s–1 and k 1964 cm–1 = 122 ± 20 s–1 at pH 5.6, and k 1945 cm–1 = 148 ± 30 s–1 and k 1964 cm–1 = 158 ± 32 s–1 at pH 8.5. The rate of decay of the heme propionate vibrations is on a time scale coincident with the rate of rebinding, suggesting that there is a coupling between ligation dynamics in the distal heme environment and the environment sensed by the heme propionates. The implications of these results with respect to the proximal His–Fe heme environment including the propionates and the positively charged or proton-donating residues in the distal pocket which are crucial for the synthesis of nitriles are discussed.
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.
The 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.
Trp283 is responsible for oxygen sensing and signal transduction in a chemotaxis signal transducer protein, Aer2.
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