Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (Fe(B)) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.
The 2.8 A crystal structure of hydroxylamine oxidoreductase of a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea, is described. Twenty-four haems lie in the centre bottom of the trimeric molecule, localized in four clusters within each monomer. The haem clusters within the trimer are aligned to form a ring that has inlet and outlet sites. The inlet is occupied by a novel haem, P460, and there are two possible outlet sites per monomer formed by paired haems lying within a cavity or cleft on the protein surface. The structure suggests pathways by which electron transfer may occur through the precisely arranged haems and provides a framework for the interpretation of previous and future biochemical and genetic observations.
Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.assembly ͉ magnetosomes ͉ prokaryote ͉ magnetic reception
A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and K m values for nitric oxide were 84 mol of nitric oxide per min/mg of protein and 0.25 M, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O 2 oxidoreductase activity with a V max of 8.4 mol of O 2 per min/mg of protein and a K m value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. (4,5,20,22), the NO reductase of the bacterium remains poorly characterized. NO, which is one of the reaction intermediates in the denitrification process (15,29), is chemically active and toxic to living cells. However, the concentration of NO in denitrifiers is stabilized only at the nano-or subnanomolar level because of the active consumption by NO reductase (12,30). NO reductases have been purified from only two kinds of denitrifying bacteria, Pseudomonas stutzeri (14, 17) and P. denitrificans ATCC 19367 (NCIB 8944) (6, 9). Both enzyme molecules contain protoheme and heme c and catalyze the reduction of NO to N 2 O in artificial electron-donating assays using phenazine methosulfate-ascorbate or 2,3,5,6-tetramethylphenylenediamine-ascorbate-horse cytochrome c. However, the physiological electron pathway to NO has not been reconstituted in vitro. Paracoccus denitrificansWe describe here a simple purification of NO reductase from P. denitrificans ATCC 35512 by using sucrose monocaprate (SM-1080) as a solubilizing detergent. The purified enzyme showed a subunit structure and spectral properties similar to those of cb-type NO reductases. Furthermore, the enzyme was very stable in the presence of the detergent and oxygen and showed not only cytochrome c-NO reductase activity but also cytochrome c-O 2 reductase activity when P. denitrificans ATCC 35512 cytochrome c-550 was used as the electron donor. We succeeded in reconstituting the electron transport from physiological cytochrome c to NO catalyzed by NO reductase. MATERIALS AND METHODSOrganisms and cultivation. P. denitrificans ATCC 35512 was anaerobically cultivated as previously described by Robertson and Kuenen (21), with some modifications. The bacterium was grown at 37ЊC in a medium containing (per liter) 1.36 g of CH 3 COONa ⅐ 3H 2 O, 3.26 g of KNO 3 , 0.8 g of K 2 HPO 4 , 0.3 g of KH 2 PO 4 , and 0.4 g of MgSO 4 and a trace amount ...
Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures.Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility. K E Y W O R D S appendage, cytoskeleton, flagella, membrane remodeling, Mollicutes, motor protein, peptidoglycan, three domains | 9Genes to Cells MIYATA eT Al.
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