Characterization of Two Type 1 Cu Sites ofHyphomicrobium denitrificansNitrite Reductase:  A New Class of Copper-Containing Nitrite Reductases

Abstract

We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization. The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs). Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively. The point mutation of the Cys ligand of each type 1 Cu with Ala gives two mutants, C114A and C260A, possessing one type 1 Cu and one type 2 Cu. The spectroscopic data of C114A reveal that the C-terminal NIR-like domain has the green type 1 Cu (type 1 Cu(C)), showing two intense absorption peaks at 455 (epsilon = 2600 M(-1) cm(-1)) and 600 nm (epsilon = 2800 M(-1) cm(-1)) and a rhombic EPR signal like those of the green type 1 Cu of Achromobacter cycloclastes NIR (AcNlR). The spectroscopic data of C260A elucidate that the N-terminal Pc-like domain in HdNIR contains the blue type 1 Cu (type 1 Cu(N)), exhibiting an intense absorption band at 605 nm (epsilon = 2900 M(-1) cm(-1)) and an axial EPR signal like those of the blue type 1 Cu of Alcaligenes xylosoxidans NIR (AxNIR). The sum of the visible absorption or EPR spectra of C114A and C260A is almost equal to the corresponding spectrum of wild-type HdNIR. The spectroscopic characterization of the type 1 Cu indicates that the geometries of the type 1 Cu(N) and Cu(C) sites are slightly distorted tetrahedral (or axially elongated bipyramidal) and flattened tetrahedral, respectively. In the cyclic voltammograms, the midpoint potentials (E(1/2)), probably because of the type 1 Cu ions of C114A and C260A, are observed at +321 and +336 mV versus normal hydrogen electrode (NHE) at pH 7.0, respectively. These values, which are close to each other, are more positive than those ( approximately +0.24-0.28 V at pH 7.0) of the type 1 Cu sites of AcNIR and AxNIR. The electron-accepting capability of C114A from cytochrome c(550) is almost similar to that of wild-type HdNIR, whereas that of C260A is very low. This suggests that the type 1 Cu(C) in the C-terminal domain is essential for the enzyme functions of HdNIR.

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii

“…H. denitrificans is characterized by its unique nitrite re- ductase, which is clearly distinct from the nitrite reductases found in other Hyphomicrobium species. It is characterized by the presence of two type I Cu sites, as opposed to the single type I Cu site found in the nitrite reductases of most bacteria (16,17,45). The role of this second type I Cu site has not been elucidated yet.…”

“…H. denitrificans (5) is commonly found in soils, freshwater environments, and activated sludge. This bacterium has the capacity to denitrify (2), and its nitrite and nitrous oxide reductases were previously characterized (15)(16)(17)(18). H. zavarzinii is a soil bacterium that has been studied mainly for the biotechnological potential of the exceptional formaldehyde dehydrogenase found in strain ZV580 (19,20).…”

Denitrifying Bacteria Isolated from Terrestrial Subsurface Sediments Exposed to Mixed-Waste Contamination

“…PCR amplicons were cleaned as described above and sequenced directly. Primers specific to the nirK gene of Hyphomicrobium denitrificans were designed using a previously published sequence of H. denitrificans strain A3151 (primer set 4 [64]). A primer set for this gene was generated using the online software Primer-BLAST at the NCBI website (51).…”

Structure and function of a hexameric copper-containing nitrite reductase

“…The amino acid sequence of the C-terminal region shows a significant similarity (48% identity) to that of class II CuNIR AniA from Neisseria gonorrhoeae (10). Moreover, according to the mutation of HdNIR, C114A and C260A possessing one type 1 Cu and one type 2 Cu demonstrated that the N-terminal and C-terminal regions have blue and green type 1 Cu sites, respectively (13,14). Here we describe the crystal structure of HdNIR at a resolution of 2.2 Å and the functions of the Cu centers.…”