A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

Lars J.C Jeuken,Fraser A Armstrong,Gerard W Canters,Martin P Verbeet,Hein J Wijma

doi:10.1074/jbc.m601610200

Lars J.C Jeuken, Fraser A Armstrong + Show 3 more

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https://doi.org/10.1074/jbc.m601610200

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Abstract

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 random-sequential 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 approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Highlights

nitrite reductase (NiR) is a homotrimer, in which each subunit contains a type-1 copper site that accepts electrons from a physiological electron donor and transfers them to a type-2 copper site that is located deeper inside the enzyme [11,12,13]
We report on investigations of the catalytic mechanism of NiR by kinetic techniques for which solutions of NiR with three different electron donors at saturating concentrations have been employed
Deviations from Henri-Michaelis-Menten Kinetics by NiR—The investigations were started by measuring the catalytic activity of nitrite reductase as a function of pH with pseudoazurin as the electron donor at saturating concentrations

Summary

Introduction

NiR is a homotrimer, in which each subunit contains a type-1 copper site that accepts electrons from a physiological electron donor and transfers them to a type-2 copper site that is located deeper inside the enzyme [11,12,13]. The type-2 copper is coordinated by three histidines and forms the active site together with a water network, an aspartate, and a histidine The latter two residues hydrogen bond to the nitrite and donate protons for the enzymatic reaction (14 –18). Among key observations indicating that nitrite binds first to the oxidized copper are that the reduced type-2 site in NiR prefers a low occupancy of the fourth coordination position [14, 21, 22] and that the prereduced NiR only sluggishly reacts with nitrite [22, 27]. The experiments have been complemented by electrochemical experiments on NiR that was immobilized on a rotating disk graphite electrode It appears that the results can be analyzed and fitted satisfactorily on the basis of a random sequential mechanism

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