No description available.

A series of macrocyclic FeIII/II and CoIII/II transition metal complexes has been selected and tested to serve as artificial mediators in redox potentiometry of proteins and also in catalytic cyclic voltammetry of redox active enzymes. The potentials of these mediators ranges approximately from +200 mV to -600 mV vs the normal hydrogen electrode (NHE) at pH 7, which spans the range of most redox active proteins. These coordination complexes are mostly stable in both the oxidized and the reduced forms and show pH-independent electrochemistry within the range 6 < pH < 9. A significant advantage of these mediators is to exhibit very weak visible absorption maxima which enable proteins with low extinction coefficients to be studied by optical potentiometry without spectral interference from the mediators. These mediators have been applied to the catalytic electrochemistry of dimethyl sulfoxide (DMSO) reductase. DMSO reductase isolated from Rhodobacter capsulatus has been studied extensively in this work. It is an 82 kDa monomeric, soluble enzyme found in the periplasmic space of the organism where it is responsible for catalyzing the reduction of DMSO to dimethyl sulfide (DMS). DMSO is a rather inert and difficult to analyse compound, which appears in foods and beverages, such as wine, coffee, and tea. Enzyme based methods offer an effective way to detect DMSO in these samples. Rhodobacter capsulatus DMSO reductase contains only a single molybdenum cofactor which cycles between MoVI and MoIV during catalysis and provides a rare example of a Mo enzyme that has no other cofactors such as hemes, Fe-S clusters and flavins. Previous studies of direct (unmediated) electrocatalysis by DMSO reductase have shown only modest activity in comparison to that of other Mo enzymes. Mediated electrochemistry of DMSO reductase from Rhodobacter capsulatus using low-potential macrocyclic complexes such as [Co(trans-diammac)]3+, [Co(cis-diammac)]3+, or [Co(AMMEsar)]3+ as a mediator has been studied. The normal transient CoIII/II voltammetric response of the complex is converted into a sigmoidal (steady state) waveform in the presence of both DMSO and DMSO reductase. A single set of enzymatic rate and equilibrium constants has been determined through digital simulation (DigiSim) of the cyclic voltammetry performed under conditions where the scan rate, DMSO concentration, DMSO reductase concentration and mediator concentration were varied systematically. This information provides new insight to the kinetics of the DMSO reductase catalytic mechanism that has never before been obtained from steady state or stopped flow kinetics studies. Of note is that lowering the thermodynamic driving force by sequentially raising the redox potential of the mediator ([Co(trans-diammac)]3+ to [Co(cis-diammac)]3+ to [Co(AMMESar)]3+) slows the enzyme-mediator electron transfer reaction in accord with Marcus theory. Finally, preparation of enantiomerically pure sulfoxides by an electrochemical enzymatic system utilizing DMSO reductase from Rhodobacter capsulatus has also been investigated. This method has been performed using the coordination complex [Co(trans-diammac)]3+ as an electron donor during bulk electrocatalysis. The results indicate that DMSO reductase from R. capsulatus prefers to catalyze the reduction of both R-MPTSO (methyl p-tolyl sulfoxide) and R-MPSO (methyl phenyl sulfoxide) over their S-enantiomers. These results have been rationalized on the basis of the published crystal structure of DMSO reductase (R. capsulatus) with DMSO bound at the active site.

A series of macrocyclic FeIII/II and CoIII/II transition metal complexes has been selected and tested to serve as artificial mediators in redox potentiometry of proteins and also in catalytic cyclic voltammetry of redox active enzymes. The potentials of these mediators ranges approximately from +200 mV to -600 mV vs the normal hydrogen electrode (NHE) at pH 7, which spans the range of most redox active proteins. These coordination complexes are mostly stable in both the oxidized and the reduced forms and show pH-independent electrochemistry within the range 6 < pH < 9. A significant advantage of these mediators is to exhibit very weak visible absorption maxima which enable proteins with low extinction coefficients to be studied by optical potentiometry without spectral interference from the mediators. These mediators have been applied to the catalytic electrochemistry of dimethyl sulfoxide (DMSO) reductase. DMSO reductase isolated from Rhodobacter capsulatus has been studied extensively in this work. It is an 82 kDa monomeric, soluble enzyme found in the periplasmic space of the organism where it is responsible for catalyzing the reduction of DMSO to dimethyl sulfide (DMS). DMSO is a rather inert and difficult to analyse compound, which appears in foods and beverages, such as wine, coffee, and tea. Enzyme based methods offer an effective way to detect DMSO in these samples. Rhodobacter capsulatus DMSO reductase contains only a single molybdenum cofactor which cycles between MoVI and MoIV during catalysis and provides a rare example of a Mo enzyme that has no other cofactors such as hemes, Fe-S clusters and flavins. Previous studies of direct (unmediated) electrocatalysis by DMSO reductase have shown only modest activity in comparison to that of other Mo enzymes. Mediated electrochemistry of DMSO reductase from Rhodobacter capsulatus using low-potential macrocyclic complexes such as [Co(trans-diammac)]3+, [Co(cis-diammac)]3+, or [Co(AMMEsar)]3+ as a mediator has been studied. The normal transient CoIII/II voltammetric response of the complex is converted into a sigmoidal (steady state) waveform in the presence of both DMSO and DMSO reductase. A single set of enzymatic rate and equilibrium constants has been determined through digital simulation (DigiSim) of the cyclic voltammetry performed under conditions where the scan rate, DMSO concentration, DMSO reductase concentration and mediator concentration were varied systematically. This information provides new insight to the kinetics of the DMSO reductase catalytic mechanism that has never before been obtained from steady state or stopped flow kinetics studies. Of note is that lowering the thermodynamic driving force by sequentially raising the redox potential of the mediator ([Co(trans-diammac)]3+ to [Co(cis-diammac)]3+ to [Co(AMMESar)]3+) slows the enzyme-mediator electron transfer reaction in accord with Marcus theory. Finally, preparation of enantiomerically pure sulfoxides by an electrochemical enzymatic system utilizing DMSO reductase from Rhodobacter capsulatus has also been investigated. This method has been performed using the coordination complex [Co(trans-diammac)]3+ as an electron donor during bulk electrocatalysis. The results indicate that DMSO reductase from R. capsulatus prefers to catalyze the reduction of both R-MPTSO (methyl p-tolyl sulfoxide) and R-MPSO (methyl phenyl sulfoxide) over their S-enantiomers. These results have been rationalized on the basis of the published crystal structure of DMSO reductase (R. capsulatus) with DMSO bound at the active site.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

No description available.