Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF)

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric alpha-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and H-1-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblasts. As we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei : Istiophoredae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa. were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation; mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

DOSY NMR studies of chemical exchange behavior of poly(2-hydroxyethyl methacrylate)

PFG-NMR was used to study the chemical exchange of linear PHEMA having a range of molecular weights with water in DMSO containing varying quantities of water. The aim was to investigate the use of PFG-NMR to study chemical exchange between a polymer with exchangeable protons and a small fast diffusing molecule to provide insight into the conformation adopted by a polymer in solution. The experimental data were simulated closely for the two-site exchange case using the Bloch equations modified for chemical exchange and diffusion. The exchange rate could be used to detect changes in polymer conformation resulting from changes in the solvent. PHEMA of MW 10 000 showed significant time-dependent changes in exchange rate, resulting from preferential solvation of the OH sites by water, and subsequent conformational changes which altered accessibility of the OH sites to water. This behavior was not observed for larger MW PHEMA, which adopted a stable conformation immediately. Large changes in the exchange rate were not reflected in changes to the hydrodynamic radius, suggesting that a minimal overall change in the chain dimensions occurred. DMSO was found to be a poor solvent for PHEMA, which adopts a compact conformation in DMSO. This work has demonstrated that PFG-NMR is a sensitive method for detecting subtle changes in polymer conformation in polymers with exchangeable protons.