In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.

Sepsis is a systemic inflammatory response syndrome to infection. Human b-defensin 1 (DEFB1) is a multifunctional mediator in infection and inflammation, which has been largely explored in ex vivo studies. The present case-control study was designed to investigate whether DEFB1 genomic variations are associated with the susceptibility to and the outcome of severe sepsis in 211 patients with severe sepsis and 157 ethnic-matched healthy controls. After correcting for multiple testing, the À44G/C was the only polymorphism found to show significant associations with both the susceptibility to and the fatal outcome of severe sepsis (P ¼ 0.0049, odd ratio (OR) 1.971 and P ¼ 0.002, OR 2.406, respectively). Haplotype À20A/À44C/À52G showed a protective role against severe sepsis (P ¼ 0.0066, OR 0.6751), whereas haplotype À20G/À44G/À52G served as a risk factor for the fatal outcome of severe sepsis (P ¼ 0.0052, OR 2.427). These findings provide further evidence that b-defensin 1 may play a role in the pathogenesis of severe sepsis.

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