Roles of four iron centers in Paracoccus halodenitrificans nitric oxide reductase

Reactions of Paracoccus halodenitrificans nitric oxide reductase (NOR) containing four iron centers, a low spin hemec, a low spin heme b, a high spin heme b and a non-heme iron, have been studied to show the roles of each iron center. Soon after reacting the resting (oxidized) NOR with L-ascorbate, the low spin heme c and low spin heme b were reduced to a considerable extent but the high spin heme b was still in the oxidized form and was reduced slowly. When CO acted on the reduced NOR, the high spin heme b center changed to a low spin state. On the other hand, when NO acted on the resting NOR, no apparent spectral change was observed. However, when NO acted on the reduced NOR (a steady state condition, excess dithionite is present), both of the low spin centers changed to be partly in the oxidized form. A small but clear new EPR signal with g = 4.1 appeared together with some new signals at the g = 2 region soon after the action of NO on the reduced NOR. During incubation at room temperature the nitrosyl-heme signal typical of 5-coordination developed. These results suggested that both the high spin-heme b center and the non-heme iron are the reaction centers and their reductions are indispensable for the enzyme process in contrast to the reaction mechanism proposed for the P-450 type NOR(P-450nor).