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Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres.

ABSTRACT: Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this 'blue' nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.

SUBMITTER: Abraham ZH

PROVIDER: S-EPMC1134921 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other

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Project description:The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major ((34)S/(32)S) and minor ((33)S/(32)S, (36)S/(32)S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in (34)S/(32)S (hereafter, [Formula: see text]) to be 15.3 ± 2‰, 2?. The accompanying minor isotope effect in (33)S, described as [Formula: see text], is calculated to be 0.5150 ± 0.0012, 2?. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3-0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in (34)?DsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of [Formula: see text] is similar to the median value of experimental observations compiled from all known published work, where (34)? r-p = 16.1‰ (r-p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments ([Formula: see text] 17.3 ± 1.5‰, 2?) and in modern marine sediments ([Formula: see text] 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern and ancient environments.

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Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres.

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