Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

Abstract

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe3+ in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be −120mV. On reduction with Na2S2O4 or Na2S2O4+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron–sulphur centre. The midpoint potential of this centre was very low, about −570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na2S2O4, the iron–sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron–sulphur centre. Other ligands to haem, such as CO and the reaction product NH3, had similar but less pronounced effects, and also changed the lineshape of the iron–sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na2S2O4 in various proportions. Signals were interpreted as due to the reduced iron–sulphur centre (with slightly different g values), a haem–NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem–NO spectrum was more intense, whereas in the presence of an excess of Na2S2O4 it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem–NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH4+.