An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes

Abstract

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.

Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket

“…A variety of kinetic mechanisms were investigated to determine whether they could account for the observed kinetic characteristics exhibited by the reaction of metmyoglobin with hydrogen peroxide. Each mechanism was explored in the time domain by using the Gear variable-step numerical integration method to generate theoretical time courses [25]. The time courses were then fitted to the experimental data by varying the rate constants in the scheme being tested.…”

A quantitative model for the mechanism of action of the cytochrome c peroxidase of Pseudomonas aeruginosa

“…Spectrophotometric measurements were made on a Cary 11 8c spectrophotometer. Rapid reactions were performed in a Gibson-Milnes stopped-flow apparatus (Gibson & Milnes, 1964), equipped with a Tracor Northern TN6500 rapid-scan spectrophotometer (Blackmore et al, 1990). Digitized experimental reaction time courses were converted into ASC II files for analysis on a NEC 386SX PC computer.…”

“…Reaction traces were analysed in terms of either singleor double-exponential processes, as appropriate, by using non-linear least-squares fitting programs described previously (Brittain, 1986). Numerical simulations of various model reaction systems were performed by using Gear numerical integration techniques (Gear, 1971;Blackmore et al, 1990), employing previously reported algorithms (Brittain, 1990).…”

Kinetics of Dithionite-Dependent Reduction of Cytochrome P450 3A4: Heterogeneity of the Enzyme Caused by Its Oligomerization

“…In particular, reduction of cytochrome P450 by dithionite is used in routine quantitative determinations of the heme protein. Studies of the kinetics of reduction by dithionite were employed to probe apparent conformational transitions and changes in the ligand sphere of the heme iron in such heme-containing enzymes as bovine heart cytochrome c oxidase ( , ), cytochrome bo from Escherichia coli ( , ), and some other biological electron carriers ( − ). For most heme proteins studied it was shown that the mechanism of reduction involves the dithionite anion monomer SO •- 2 as the reducing agent ( − ).…”