Abstract
Nitrous oxide reductase from Wolinellasuccino- genes, an enzyme containing one heme c and four Cu atoms/subunitofM,=88,000,wasstudiedbyelectron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with g, = 3.10 and an axial Cu reso- nance was observed with g,,= 2.17 andg.= 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the glland g, regions with average sepa- rations of 45.2 and 26.2 gauss, respectively. The hy- perfine components are attributed to Cu(1)-Cu(I1)S = 1h (half-met) centers. Reductionof the enzyme with dithionitecausedsignalsattributableto hemecandCu to disappear; exposure of that sample to NzO for a few min caused the reappearanceof the g = 3 . 1 0 component and a new Cu signal with g,,= 2.17 and g, = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence fora Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20%of the total Cu, respectively, orabout one spinlsubunit.
The enzyme offers the first example of a nitrous oxide reductase which can havetwostates of high activity that present very different EPR spectra of Cu. These two statesmay represent enzyme in two differ- ent stagesof the catalytic cycle.