Nitrite Reductase from Achromobacter fischeri: Molecular Weight and Subunit Structure

Abstract

The Achromobacter fischeri nitrite reductase used in the present studies was monodisperse as judged by ultracentrifugation and disc-gel electrophoresis. The native enzyme has an average molecular weight of 80000 as determined by the Archibald approach-to-equilibrium method, disc-gel electrophoresis and from a combination of hydrodynamic properties. The sedimentation and diffusion coefficients are 5.25 S and 60.5 p.m2 s-l respectively, frictional ratio ( f / f o ) 1.25, and Stokes' radius 3.49 nm.Nitrite reductase does not dissociate in the presence of 6 M guanidine-HC1 or 6 M urea.The enzyme however splits into two physically indistingiushable subunits upon treatment with 6 M guanidine-HC1 in combination with lo/,, 2-mercaptoethanol or 101, sodium dodecylsulfate and 1 2-mercaptoethanol. In both the systems the subunit molecular weight has been found to be approximately 39000. Methionine is the sole N-terminal residue detected. These results indicate that Achromobacter nitrite reductase is composed of two subunits of equivalent size which are covalently bonded by a disulfi.de bridge.

A reappraisal of the role of the low potential c-type cytochrome (cytochrome c-552) in NADH-dependent nitrite reduction and its relationship with a co-purified NADH oxidase in Escherichia coli K-12

“…This pathway is similar to the one proposed by Prakash and Sadana [IS] for nitrite reduction in Achromobacter ~s~~~ where the nitrite reductase is also a high molecular weight c-type cytochrome (M 80 000) but composed of two identical subunits each containing one heme group [16]. Based on the activity data and the spectral study, we proposed an electron transport pathway from NADH to nitrite as follows: reduced methyl viologen where FAD either is part of the NADH oxidase or serves as a free electron carrier.…”

Metabolic role and properties of nitrite reductase of nitrate-ammonifying marine Vibrio species

“…In marine sediments, species of the Vibrio group constitute the major part of the nitrate-'ammonifying' bacterial flora [1,4]. The terminal enzyme of nitrate ammonification, i.e., nitrite reductase, has been purified from Achromobacterfischeri (an organism later reclassified as Vibrio fischeri) and shown to be a heme protein with M r 80 000 and two c-type heme groups per molecule [5]. Since nitrite reductases of nitrate ammonifying bacteria seem rather diverse in structure and cofactor content [6][7][8], it was of interest to study the properties of the enzyme from another Vibrio species.…”

Wolinella succinogenes nitrite reductase: Purification and properties

“…It should be mentioned that assimilatory and dissimilatory sulfite reductases are fortuitous nitrite reductases, and such activity has been attributed [18,19] a Specific activity is calculated as #mol of nitrite converted to ammonia/min/mg purified nitrite reductase b Calculated from turnover number of purified enzyme based on 6 mol of reduced benzyl viologen oxidized/mol of nitrite converted into ammonia c Expressed as number of heme c groups/molecule of protein to the intrinsic capability of siroheme-containing proteins to catalyze 6-electron reduction reactions [21 ]. These reductases and the dissimilatory nitrite reductases are known to be able to reduce hydroxylamine (excluded as an intermediate during nitrite reduction) to ammonia.…”