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FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y.(2013) J. Phys. Chem. B117, 15786−15791].

The crystal structure of the heme-binding domain of the bacterial O 2 sensor protein FixL from Rhizobium meliloti (RmFixL) was determined at 1.4-A˚ resolution. Combining the structural data with some spectroscopic, biochemical, and molecular biological data, we discussed the O 2 sensing mechanism by FixL. We proposed that the interaction of the iron-bound O 2 with its surroundings would be a key step in triggering the intramolecular signal transduction from the O 2 -binding heme domain to the catalytic histidine kinase domain.

Nitric oxide reductase (NOR) isolated from Fusarium oxysporum catalyzes the reduction reaction of nitric oxide (NO) to nitrous oxide (N 2 O). In this reaction, the transient reaction intermediate ( t =∼20 s) is produced by the reduction of ferric-NO complex with NADH. To elucidate the electronic structure of the intermediate, we measured its resonance Raman spectrum using the continuous-flow method. From these spectral features, we have concluded that the electronic structure of the intermediate in the NO reduction reaction by fungal NOR is FeNOH. Further, we have found that the Ser286/Asp393 hydrogen-bond network is the proton pathway to the active site on the basis of crystallographic and mutagenesis studies. From these results, we discuss the molecular mechanism of NO reduction reaction catalyzed by fungal NOR.

The high-molecular-weight (HMW) glutenin subunit composition of seed storage protein of 174 Japanese hexaploid wheat (Triticum aestivum) landraces have been examined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis system. Twenty four different, major glutenin HMW subunits were identified, and each of the landraces contained three to five subunits and 17 different glutenin subunit patterns were observed for 13 alleles in the landraces. On the basis of HMW glutenin subunits composition, Japanese landraces showed a specific allelic variation, close to Japanese commercial wheats in HMW glutenin subunits, different from those in alien hexaploid wheats. Further, it could be concluded that all common glutenin alleles can be found in the 174 landraces originated from Japan. The variation detected in the glutenin subunits is useful for variety identification, has a bearing on our understanding of hexaploid wheat genetic resource evolution in Japan, and raises questions concerning the nature of this genetic variation.

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels onto sequencer stable supports for protein microsequence analysis in a gas-phase sequencer. Using this technique, I determined the amino acid sequences of the endosperm proteins in Japanese hexaploid commercial wheats (Triticum aestivum). Based on sequence determination of the Japanese hexaploid wheats, the endosperm protein could be easily characterized. Wheat endosperm protein, extracted in the presence of 2-mercaptoethanol and SDS, fractionated into many protein polypeptides using 2D-PAGE under dissociating conditions. These components were grouped into HMW glutenin subunits, a-, ß- or ?-gliadins, and novel protein polypeptides by using the N-terminal amino acid sequences. The novel endosperm protein polypeptides were detected, and two new types of N-terminal amino acid sequences have been found for protein poly-peptides. These polypeptides have much faster electrophoresis mobility during 2D-PAGE and are therefore probably a much smaller size than any other peptides of endosperm protein groups found in hexaploid wheat. Ten protein polypeptides have been purified from cultivars of Japanese wheat. Some differences in the contents of amino acids for four protein polypeptide spots were apparent in Japanese wheat.

FixL of Rhizobium meliloti(RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O2levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 Å resolution. Based on the structural and spectroscopic analyses, we propose the O2sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O2and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O2and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O2and Fe-CN stretching frequencies were shifted from 575 to 570 cm−1(Fe-O2), and 504 to 499 cm−1, respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O2sensing activity was destroyed, thus confirming our proposed mechanism.

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the reduction of NO to N2O through its ferric-NO complex, the first intermediate of the catalysis. Crystal structures of the ferric-NO forms of wild type (WT) fungal NOR, and of the Ser286→ Val and Ser286→ Thr mutant enzymes were determined to 1.7-Å resolution at cryogenic temperature (100 K). This shows a slightly tilted and bent NO binding to the heme iron, in sharp contrast to the highly bent NO coordination found in ferrous hemoproteins. In the WT structure, a specific hydrogen-bonding network that connects the active site to the solvent was identified, H2O(Wat74)-Ser286-H2O(Wat33)-Asp393-solvent. Wat74is located 3.10 Å from the iron-bound NO. Replacement of Ser286with Val or Thr scarcely alters the NO coordination structure but expels the water molecules, Wat74from the active site. The Asp393mutation does not influence the position of Wat74, but disrupts the hydrogen-bonding network at Wat33, as evidenced by enzymatic, kinetic, and spectroscopic (resonance Raman and IR) results. The structural changes observed upon the Ser286or the Asp393mutation are consistent with the dramatic loss of the enzymatic activity for the NO reduction of fungal NOR. We have conclusively identified the water molecule, Wat74, adjacent to the iron-bound NO as a proton donor to the Fe-NO moiety. In addition, we find the hydrogen-bonding network, H2O(Wat74)-Ser286-H2O(Wat33)-Asp393, as a proton delivery pathway in the NO reduction reaction by fungal NOR.

FixL is a transmitter protein in a two‐component system which acts as an oxygen sensor when the symbiotic Rhizobiaresides in root nodules of host plants. The oxygen‐sensor domain of Rhizobium melilotiFixL (RmFixLH) was purified by His‐tag affinity and isoelectronic focusing chromatographies, without the use of gel‐filtration chromatography. Dynamic light‐scattering measurements of RmFixLH thus obtained revealed it to be monodispersive and present as a homodimer in solution. A single crystal of RmFixLH in the met (Fe3+) form was grown in 100 mMacetic acid/NaOH buffer at pH 4.6 in the presence of 200 mMammonium acetate, using 40%(w/­v) PEG 4000 as a precipitant. A crystal of the ferrous CO form of RmFixLH was also prepared by reduction of the met crystal with Na2S2O4in an atmosphere of CO. The crystals (0.2 × 0.05 × 0.01 mm) belong to the monoclinic system (C2) with unit‐cell parameters a= 60.94, b= 37.44, c= 54.14 Å, β = 115.29° and diffract X‐rays to 1.7 Å resolution at station BL44B2 of SPring‐8, Japan. Bijvoet difference Patterson maps show a clear peak corresponding to the haem iron in RmFixLH.

FixL is a heme-based O2sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium melilotiFixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe2+, Fe2+O2, Fe2+CO, Fe3+, Fe3+F−, and Fe3+CN−states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain ofBradyrhizobium japonicumFixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A.95, 15177–15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O2sensing.

Seed storage proteins of 131 Japanese Norin wheat (Triticum aestivum) varieties were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis to determine allelic make-up in varieties at each of three loci that control high-molecular-weight (HMW) glutenin subunits. Three alleles were identified at the Glu-A1 locus, six at the Glu-B1 locus and five at the Glu-D1 locus. Twenty-four different, major glutenin HMW subunits were identified and each contained three to five subunits and seventeen different glutenin subunit patterns were observed for 19 subunits in the 131 Japanese Norin varieties. Fourteen alleles were identified by comparison of subunit mobility with that previously found in hexaploid wheat. Japanese Norin varieties showed a specific pattern of allelic variation in glutenin HMW subunits, different from that of Chinese and other country common wheats in allelic frequency at Glu-1 loci.

A Cu B-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of Cu Bseems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that Cu Bplays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.

Rhizobial FixL is a novel heme protein, which senses environmental oxygen tension and directs signal transduction via protein phosphotransfer. To identify the essential residues for heme assembly inRhizobium melilotiFixL, we individually replaced the 18 invariant hydrophobic amino acid residues (F, I, L, and V) in the heme-containing domain with alanine and histidine. Spectroscopic measurements of the soluble fractions fromfixLrecombinantEscherichia colirevealed that V152, F162, F170, I172, L185, F226, L230, and F243 as well as the proximal ligand H194 were indispensable for heme assembly. Autoxidation rates of purified I209H, I210A, and I210H were 65-fold, 15-fold, and 15-fold, respectively, faster than that of the wild type, although they retained heme in the protein. The absorption peak in the Soret region of the ferric I209H or I210H was red-shifted, suggesting that the ferric heme is a hexa-coordinate form in these mutants.

The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b561 and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b561 with those of other bacterial b-type cytochromes was observed.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 Å resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

A CuB‐deficient mutant of the Escherichia coli bo‐type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o‐to‐ligand electron transfer. Thus, the lack of CuBseems to have endowed the mutant enzyme with myoglobin‐like properties, thereby stabilizing the CO‐bound form, too. Accordingly we conclude that CuBplays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme‐copper binuclear center.

Combined experimental and modeling techniques reveal the mechanism of signal relay in the oxygen-sensing FixL-FixJ two-component system.

Variation in the electrophoretic banding patterns of high-molecular-weight (HMW) glutenin subunits of 274hexaploid wheat (Triticum aestivum) varieties from China was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 27 different major HMW glutenin subunits were identified. Each variety contained three to five subunits and 29different glutenin subunit patterns were observed in274 Chinese hexaploid wheats. Seventeen alleles were identified based on the comparison of subunits mobility with that previously identified in a set of standard hexaploid wheats. The Chinese hexaploid wheats exhibited allelic variation in HMW glutenin subunit composition and the variation differed from that of Japanese and hexaploid wheats of other countries.