Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans

Abstract

A new and relatively simple procedure to purify NO reductase from Paracoccus denitrificans by using the detergent lauryl maltoside has been developed. The purified enzyme consists of two subunits according to SDS polyacrylamide gel electrophoresis. Analysis of the content of prosthetic groups indicates the presence of non-haem iron in addition to the presence b and c cytochromes yielding a stoichiometry of haem b/haem c/non-haem iron=2:1:1. The optical spectrum of reduced NO reductase shows bands of low-spin haem c and haem b with α-band absorbance maxima at 551 nm and 558 nm, respectively. The optical spectrum of oxidized NO reductase shows a broad absorbance band around 590 nm which disappears upon reduction. This latter absorbance is ascribed to a high-spin haem b (charge-transfer) transition. The presence of high-spin haem b is also indicated by the shifts observed in the optical spectrum of oxidized NO reductase in the presence of NO or in the spectrum of reduced enzyme after addition of CO. The main features of the EPR spectrum of the oxidized enzyme are resonances from a highly anisotropic low-spin haem b ( g z =3.53) and from an anisotropic low-spin haem c with g z,y,x =2.99, 2.28, 1.46, the two haems being present in an approximate 1:1 stoichiometry. Minor signals representing about 1% of the enzyme concentration due to high-spin haem b ( g=5.8–6.2) and a novel type of signal with g=2.009 ascribed to high-spin non-haem ferric iron were also observed. The analysis of steady-state kinetic measurements of the NO reductase activity shows a sigmoidal relation between rate of NO reduction and NO concentration, consistent with a model describing sequential binding of two molecules of NO to the reduced enzyme. At high NO concentrations substrate inhibition occurs ( K i(apparent)=13.5 μM) suggested to be due to binding of NO to oxidized enzyme. The absence from the EPR spectrum of signals originating from ferric non-haem iron and ferric high-spin haem b in stoichiometric amounts with respect to the enzyme concentration is suggested to be due to an antiferromagnetic coupling between these two centers. The steady-state kinetic behaviour and the optical and EPR spectroscopic properties of the NO reductase are incorporated into a tentative structural and mechanistic model.