A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

Resonance Raman spectra of the molybdenum containing aldehyde oxidoreductase from Desulfovibrio gigas were recorded at liquid nitrogen temperature with various excitation wavelengths. The spectra indicate that all the iron atoms are organised in [2Fe-2S] type centers consistent with cysteine ligations. No vibrational modes involving molybdenum could be clearly identified. The features between 280 and 420 cm-1 are similar but different from those of typical plant ferredoxin-like [2Fe-2S] cluster. The data are consistent with the presence of a plant ferredoxin-like cluster (center I) and a unique [2Fe-2S] cluster (center II), as suggested by other spectroscopic studies. The Raman features of center II are different from those of other [2Fe-2S] clusters in proteins. In addition, a strong peak at ca. 683 cm-1, which is not present in other [2Fe-2S] clusters in proteins, was observed with purple excitation (406.7-413.1 nm). The peak is assigned to enhanced cysteinyl C-S stretching in center II, suggesting a novel geometry for this center.