An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.
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.
Blackmore
R S
RS
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Brittain
T
T
Greenwood
C
C
D000693:Anaerobiosis; D001438:Bacteroidaceae / Q000201:enzymology*; D002248:Carbon Monoxide / Q000378:metabolism; D004227:Dithionite / Q000378:metabolism; D007700:Kinetics; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D004227:Dithionite; D002248:Carbon Monoxide; D009572:Nitrite Reductases