8.
When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO
inf2
sup-
× (mg protein × min)
-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme
c molecules/M
r 58 kDa. The absorption spectrum was typical for
c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from
Desulfovibrio desulfuricans, Wolinella succinogenes and
Escherichia coli.Abbreviations DNRA
dissimilatory nitrate reduction to ammonia
- EPR
electron paramagnetic resonance
- PAGE
polyacrylamide gel electrophoresis
- NaP
i
sodium phosphate
- SDS
sodium dodecylsulfate
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