Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli

Abstract

The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.

FIG. 1.

Determination of K_m for NH₂OH. The assay procedure and assay solution for these experiments are detailed in Materials and Methods. Assays were performed at pH 7.5 (A) and 9.0 (B). The estimated K_m values for NH₂OH are 38.9 ± 3.53 mM at pH 7.5 and 2.5 ± 0.36 mM at pH 9.0.

FIG. 2.

Effect of pH on the hydroxylamine reductase activity of HCP. It is shown that the hydroxylamine reductase activity varies dramatically at various pHs. Measured activities ranged from 92 ± 19 (pH 7.0) to 458 ± 19 μmol of NH₂OH reduced min⁻¹ mg of protein⁻¹ (pH 9.0).

FIG. 3.

UV-visible absorption spectra of oxidized and reduced forms of the HCP. Experimental procedures are described in Materials and Methods. Line A, DTH-reduced form of the enzyme; line B, enzyme oxidized by addition of NH₂OH; line C, thionin-oxidized form of the enzyme. The peak in the 600-nm region is caused by the presence of oxidized thionin in the sample.

FIG. 4.

Effect of hydroxylamine reductase activity of HCP in the presence of cyanide and CO. The assay of hydroxylamine reductase activity in the presence of effectors (CN⁻ and CO) is described in Materials and Methods. Assays were performed in the presence of CN⁻ only (⧫) and CO plus CN⁻ (▴).

FIG. 5.

Effect of incubation with cyanide and CO on the hydroxylamine reductase activity of the HCP. The treatment of effectors CN⁻ and CO is described in Materials and Methods. (A) Effect of CN⁻ treatment both in the presence (□) and absence (•) of incubation with CO prior to CN⁻ treatment. (B) Enhancement the region of panel A in which CN⁻ activation occurs, in order to properly demonstrate the CN⁻ activation effect on activity.

FIG. 6.

Effect of O₂ exposure on hydroxylamine reductase activity of HCP. The exposure of HCP to O₂ and subsequent assays for hydroxylamine reductase activity are described in Materials and Methods. Shown is the effect, over time, of exposure to O₂ on the hydroxylamine reductase activity of HCP.