Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase (original) (raw)
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Journal of Biological Chemistry, 2006
The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a randomsequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO 2 ؊ ] reduction of the type-2 site precedes nitrite binding, at high [NO 2 ؊ ] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an ϳ50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism. Copper-containing nitrite reductase (NiR) 3 is one of the enzymes of the denitrification pathway (1). Denitrification globally recycles fixed nitrogen (NO 3 Ϫ , NO 2 Ϫ) to the atmosphere (N 2) with NO and N 2 O being
Protein Film Voltammetry of Copper-Containing Nitrite Reductase Reveals Reversible Inactivation
Journal of the American Chemical Society, 2007
The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of ∼0.1 s-1. The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.