Mössbauer and EPR studies on nitrite reductase from Thiobacillus denitrificans.

We have studied nitrite reductase (cytochrome cd) from Thiobacillus denitrificuns with Mossbauer, EPR, and optical spectroscopy and biochemical techniques.The molecular weight of the purified enzyme was determined by the sedimentation equilibrium method to be 118,000.Each molecule consists of two subunits with similar molecular weight of about 65,000.From the alkaline hemochrome spectra, we found two heme c and two heme d moieties per molecule.EPR and Mossbauer data provide further evidence for a 1:l heme c to heme d ratio.In the oxidized form, both hemes are low spin ferric ( S = 1/2) and yield characteristic EPR and Mossbauer spectra.The d heme exhibits EPR resonances at g = 2.50, 2.43, and 1.70 similar to those reported for chlorin di-imidazole complexes.The c heme exhibits an EPR resonance at g 3.60 suggesting a nitrogenous sixth ligand.The Mossbauer parameters, A E Q = (2.22 +: 0.02) mm/s and 6 = (0.22 2 0.02) mm/s for the c heme and aE, = (1.66 +: 0.02) mm/s and 6 = (0.20 & 0.02) mm/s for the d heme, are typical for low spin ferric complexes.The low temperature Mossbauer spectra have been evaluated in the spin Hamiltonian formalism.It was found that the ligand field model commonly used to describe low spin ferric hemes does not account properly for the observed magnetic hyperfine interactions of the heme d iron.In the reduced form, both hemes are in the ferrous states.The observed Mossbauer parameters are A E , = (1.14 & 0.04) mm/s, S = (0.45 rt 0.04) mm/s for the c heme and A& = (2.24& 0.04) mm/s, 6 = (0.97 2 0.04) mm/s for the d heme.This observation demonstrates that, upon reduetion, the c heme remains low spin ( S = 0) and the d heme becomes high spin ( S = 2).Nitrite reductase (cytochrome cd) from Thiobacillus denitrificans is a terminal respiratory enzyme (1, 2).Its biological function is the reduction of nitrite (NOz-) to nitric oxide (NO), a major pathway in the dissimilatory nitrate metabolism.Aside from its physiological function as a nitrite reducing agent, nitrite reductase also has the ability to utilize oxygen