Purification of two nitrate reductases from Xanthomonas maltophilia grown in aerobic cultures

Abstract

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A615-630 indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 μmol of nitrite formed per min per mg of protein. The cytochrome- containing NR(I) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa α-subunit, a 64-kDa β-subunit, and a 23-kDa γ-subunit with relative band intensities indicative of a 1:1:1 α/β/γ subunit ratio and a M(r) of 222,000. The electronic spectrum of dithionite- reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.