S. Bailey

Molybdenum Active Centre of Dmso Reductase from Rhodobacter Capsulatus: Crystal Structure of the Oxidised Enzyme at 1.82-A Resolution and the Dithionite-Reduced Enzyme at 2.8-A Resolution

Abstract The 1.82-Å X-ray crystal structure of the oxidised (Mo(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-Å resolution, to an R factor of 14.8% (Rfree = 18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4 Å or 2.5 Å, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-Å structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-Å resolution, has also been obtained from crystals soaked in mother liquor buffered at pH 7.0. No differences are observed in the structure at pH 7 when compared with the native crystal structure at pH 5.5.

The crystal and molecular structures of diferric porcine and rabbit serum transferrins at resolutions of 2.15 and 2.60 Å, respectively

The serum transferrins are monomeric proteins with a molecular mass of around 80 kDa and are responsible for the transport of iron in vertebrates. The three-dimensional structures of diferric porcine and rabbit serum transferrin have been refined against X-ray diffraction data extending to 2.15 and 2.60 A, respectively. Data for both proteins were collected using synchrotron radiation at temperatures of 277 K. The porcine protein crystallizes in the space group C2, with unit-cell parameters a = 223.8, b = 44.9, c = 78.9 A, beta = 105.4 degrees with one molecule in the asymmetric unit. The structure was solved by molecular-replacement methods using rabbit serum transferrin as the search model. The structure was refined using REFMAC, with a final residual of 13.8% (R(free) = 18.2% for a 5% data sample) for all data to 2.15 A. The final model comprises 5254 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, one N-acetyl glucosamine moiety and 494 water molecules. The rabbit protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = 127.2, c = 144.9 A and one molecule per asymmetric unit. The structure was solved using the method of multiple isomorphous replacement and refined using REFMAC to give a final residual of 18.6% (R(free) = 22.2% for a 5% data sample) for all data to 2.60 A. The final model comprises 5216 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, a Cl(-) anion and 206 solvent molecules; there is no clear indication of the carbohydrate moiety attached to Asn490 (rabbit serum numbering). Both molecules adopt a bilobal structure typical for members of the transferrin family. Each of the structurally homologous lobes contains two dissimilar domains with a single iron-binding site buried within the interdomain cleft. The porcine serum protein lacks an interdomain disulfide bridge close to the connecting peptide between the lobes, but this seems to have little effect on the overall orientation of the lobes. The N-lobes of both proteins possess lysine residues, one from each of the two domains, that lie in close proximity to one another to form the so-called dilysine trigger. The more acid-labile release of iron from serum transferrins than from lactoferrins is discussed.

Molecular basis for enzymatic sulfite oxidation: How three conserved active site residues shape enzyme activity

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (∼60 and 200 s–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus

The dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus is known to retain its three‐dimensional structure and enzymatic activity upon substitution of molybdenum, the metal that occurs naturally at the active site, by tungsten. The redox properties of tungsten‐substituted DMSOR (W‐DMSOR) have been investigated by a dye‐mediated reductive titration with the concentration of the WV state monitored by EPR spectroscopy. At pH 7.0, E m(WVI/WV) is −194 mV and E m(WV/WIV) is −134 mV. Each E m value of W‐DMSOR is significantly lower (220 and 334 mV, respectively) than that of the corresponding couple of Mo‐DMSOR. These redox potentials are consistent with the ability of Mo‐DMSOR to catalyze both the reduction of DMSO to DMS and the back reaction, whereas W‐DMSOR is very effective in catalyzing the forward reaction, but shows no ability to catalyze the oxidation of DMS to DMSO.

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Abstract Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (MoVI) protein has been investigated in solution at 77 K; the Mo K-edge position (20006.4 eV) is consistent with the presence of MoVI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71 Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32 Å and two at 2.47 Å, and two O atoms, one at 1.92 Å (considered to be H-bonded to Trp 116) and one at 2.27 Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (MoVI) DMSO reductase at 77 K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (MoVI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4 eV and 20004.5 eV, respectively; these values, together with the lack of a significant presence of MoV in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of MoIV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05 Å and one at 2.51 Å, and four S atoms at 2.36 Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69 Å, one at 1.91 Å and one at 2.11 Å, plus four S atoms, two at 2.28 Å and two at 2.37 Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to MoIV with an Mo-O bond of length 1.92 Å.

Crystallization and preliminary X-ray analysis of sulfite dehydrogenase from Starkeya novella

Crystals of purified heterodimeric sulfite dehydrogenase from Starkeya novella have been grown using vapour diffusion. X-ray diffraction data have been collected from crystals of the native protein at lambda = 1.0 A and close to the iron absorption edge at lambda = 1.737 A. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 97.5, b = 92.5, c = 55.9 A. Native data have been recorded to 1.8 A resolution and Fe-edge data to 2.5 A.

In Vivo Oxo Transfer: Reactions of Native and W-Substituted Dimethyl Sulfoxide Reductase Monitored by 1H NMR Spectroscopy

Molybdenum enzymes catalyze a wide variety of reactions that involve the net transfer of an oxygen atom either to or from the substrate with the metal cycling between the oxidation states Mo and Mo. The periplasmic dimethyl sulfoxide reductases (DMSORs) of the photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides function in a respiratory chain with DMSO as the terminal electron acceptor. The DMSORs catalyze the environmentally important reaction (1) 3] that involves the direct transfer of an oxygen atom from DMSO to Mo producing dimethyl sulfide (DMS).

Preliminary crystallographic studies of dimethylsulfoxide reductase from Rhodobacter capsulatus.

Dimethylsulfoxide reductase from the photosynthetic bacterium Rhodobacter capsulatus has been crystallized in two similar forms which are suitable for X-ray structure determination. Both crystals forms belong to space group P4(1)22 or P4(3)22, with cell dimensions a = b = 80.81, c = 229.75 A (type I crystals) or a = b = 89.30, c = 230.05 A (type II crystals) and one molecule in the asymmetric unit. Diffraction has been observed to at least 2.0 A in type I crystals and to 2.6 A in type II crystals. Dimethylsulfoxide reductase from Rhodobacter is the simplest molybdenum oxotransferase known and this makes it an ideal model to study the structure and function of this class of enzymes.

ALSHub: How Users Access the Advanced Light Source

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Photon Science at the ALS for Sustainable Energy

Our current fossil-fuel-based economy is causing potentially catastrophic changes to our planet. The quest for renewable, nonpolluting sources of energy requires us to understand, predict, and ultimately control matter and energy at the electronic, atomic, and molecular levels.