Sakurai T, Sakurai N, Matsumoto H, Hirota S, Yamauchi O
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan.
Biochem Biophys Res Commun. 1998 Oct 9;251(1):248-51. doi: 10.1006/bbrc.1998.9451.
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).
对含四个铁中心（一个低自旋血红素 c、一个低自旋血红素 b、一个高自旋血红素 b 和一个非血红素铁）的嗜盐反硝化副球菌一氧化氮还原酶（NOR）的反应进行了研究，以揭示每个铁中心的作用。在将静止（氧化态）的 NOR 与 L-抗坏血酸反应后不久，低自旋血红素 c 和低自旋血红素 b 被大量还原，但高自旋血红素 b 仍处于氧化态且还原缓慢。当 CO 作用于还原态的 NOR 时，高自旋血红素 b 中心转变为低自旋态。另一方面，当 NO 作用于静止的 NOR 时，未观察到明显的光谱变化。然而，当 NO 作用于还原态的 NOR（稳态条件，存在过量连二亚硫酸盐）时，两个低自旋中心都部分转变为氧化态。在 NO 作用于还原态的 NOR 后不久，在 g = 4.1 处出现了一个小但清晰的新 EPR 信号，同时在 g = 2 区域出现了一些新信号。在室温孵育期间，出现了典型的 5-配位亚硝酰血红素信号。这些结果表明，高自旋血红素 b 中心和非血红素铁都是反应中心，与 P-450 型 NOR（P-450nor）提出的反应机制相反，它们的还原对于酶促过程是必不可少的。
