Prosthetic group content and ligand binding properties of a spinach catalase

The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach

“…The difference spectrum for the complex formed by the recombinant enzyme with nitrite ( Fig. 3A), which exhibits maxima at 424 and 560 nm, a shoulder at 528 nm and minima at 380, 504, 592 and 696 nm, is very similar to, but not identical with, that previously obtained for nitrite reductase isolated from spinach leaf (Hirasawa et al 1987). In contrast, the difference spectrum for the complex formed by the recombinant enzyme with ferredoxin (Fig.…”

“…Chemical modification of nitrite reductase tryptophan residues by treatment with N-bromosuccinimide (NBS) was carried out as described previously (Hirasawa et al 1994b). Dissociation constants (K d ) for the binding of nitrite (Hirasawa et al 1987) and the binding of ferredoxin (Hirasawa and Knaff 1985) to nitrite reductase were calculated from spectral perturbation measurements, carried out as described previously. Total iron (Miller and Massey 1965), siroheme (Siegel et al 1973) and acid-labile sulfide (Siegel et al 1973) contents were measured using standard methods.…”

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production

“…The absorbance maxima (421 nm and 564 nm) and minima (379 nm, 586 nm and 693 nm) are similar to those reported previously for the nitrite reductase isolated from C. reinhardtii cells (Romero et al 1987) and those reported for other ferredoxin-dependent nitrite reductases (Vega and Kamin 1977; Hirasawa and Tamura 1980; Hirasawa et al 1987; Mikami and Ids 1989; Bellissimo and Privalle 1995; Kuznetsova et al 2004b). A plot of the absorbance difference at 421 nm minus 379 nm arising from formation of this complex vs. nitrite concentration (not shown) were hyperbolic, consistent with the presence of a single binding site with a K d value of 50 µM.…”

“…Its spectrum is quite similar to that reported previously for the enzyme isolated directly from C. reinhardtii cells (Romero et al 1987) and those of other ferredoxin-dependent, nitrite reductases (Zumft 1972; Hucklesby et al 1976; Vega and Kamin 1977; Hirasawa and Tamura 1980; Ida and Mikami 1986; Ida et al 1989; Ip et al 1990; Bellissimo and Privalle 1995; Curdt et al 2000; Vigara et al 2002; Tripathy et al 2007) and suggested that, like other nitrite reductases (Hase et al 2006), it contains a siroheme and a [4Fe-4S] cluster as prosthetic groups. EPR spectra (Figure 2) of the as-isolated, oxidized enzyme, which contains a high-spin ( S = 5/2) Fe 3+ siroheme and an EPR-silent ( S = 0) [4Fe-4S] 2+ cluster (Aparicio et al 1975; Stoller et al 1977; Cammack et al 1978; Lancaster et al 1979; Ip et al 1990; Kuznetsova et al 2004a; Kuznetsova et al 2004b; Tripathy et al 2007) and of its cyanide adduct, in which the [4Fe-4S] cluster remains in the oxidized 2 + redox state and the high spin ( S = 5/2) Fe 3+ siroheme has been converted to a low-spin ( S =1/2) Fe 3+ siroheme cyanide complex (Aparicio et al 1975; Cammack et al 1978; Lancaster et al 1979; Wilkerson et al 1983; Hirasawa et al 1987; Kuznetsova et al 2004b), confirm the presence of the siroheme, as does pyridine hemochromagen analysis of an acidic-acetone extract of the protein. As is also- shown in Figure 2, addition of dithionite to the cyanide-adduct, reduces both prosthetic groups, resulting in an EPR-silent low-spin ( S = 0) Fe 2+ siroheme and the characteristic spectrum of a magnetically isolated ( S = 1/2) reduced [4Fe-4S] 1+ cluster (Ip et al 1990; Aparicio et al 1975; Stoller et al 1977; Cammack et al 1978; Lancaster et al 1979; Wilkerson et al 1983; Kuznetsova et al 2004a).…”

Remembering David B. Knaff (1941–2016)

“…Over the long period of his research career, he published over 20 papers on this enzyme, using a wide variety of biochemical, biophysical, and molecular biological techniques in this work. David's first two papers on nitrite reductase dealt with the presence and properties of the two prosthetic groups in the enzyme: the siroheme and the iron-sulfur center (Hirasawa et al 1987). Midpoint potentials were determined for both groups and a value of -50 mV was found for the siroheme and an estimated value of -550 mV was found for the iron-sulfur cluster.…”