INACTIVATION OF FORMATE DEHYDROGENASE FROM METHANOBACTERIUM-FORMICICUM BY CYANIDE
INACTIVATION OF FORMATE DEHYDROGENASE FROM METHANOBACTERIUM-FORMICICUM BY CYANIDE
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DOI：
10.1021/bi00373a004
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发表时间：
1986-12-16
期刊：
影响因子：
2.9
通讯作者：
FERRY, JG
中科院分区：
文献类型：
--
作者：
BARBER, MJ;MAY, HD;FERRY, JG
甲酸甲烷杆菌的甲酸脱氢酶含有Mo、FAD和Fe/S簇的修复基团。用甲酸盐或二亚硫酸盐部分还原天然酶产生了Mo(V)电子顺磁共振信号，G_1=2.020，G_2=2.006，G_3=1.997，表现出近各向同性的超精细相互作用为两个等价的强耦合质子(A_1=0.45，A_2=0.55，A_3=0.5mT)，经2H取代鉴定。氧化甲酸脱氢酶与氰化物孵育后，酶活性发生不可逆性损失，硫化物处理可使酶活性恢复。氰化物处理的甲酸脱氢酶释放等摩尔硫氰酸盐，表明钼失去了一个末端的硫配体。氰化物灭活氧化的天然酶导致Mo(V)电子顺磁共振信号向高场移动(G1=2.005，G2=1.998，G3=1.989)，超精细分裂模式的复杂性降低，这是由于失去了一个强耦合、可交换的质子，剩余的耦合质子产生的耦合常数分别为A1=1.0 5、A2=-.85和A3=0.88mT。此外，氰化物处理改变了酶电位滴定过程中Mo(V)EPR信号的行为，使Mo(VI)/Mo(V)和Mo(V)/Mo(IV)氧化还原对的中点电位分别为-319 mV和-321 mV。对天然和氰化物处理的甲酸脱氢酶的光谱和热力学性质的比较表明，失活模式与相关酶、黄嘌呤、氧化酶的失活模式相似，导致硫残基的损失，产生甲酸脱氢酶的“脱硫型”。
Formate dehydrogenase from Methanobacterium formicicum contains Mo, FAD, and Fe/S clusters prosthetic groups. Partial reduction of the native enzyme with either formate or dithionite yielded a Mo(V) electron paramagnetic resonance (EPR) signal, g1 = 2.020, g2 = 2.006, and g3 = 1.997, exhibiting nearly isotropic superhyperfine interaction to two, equivalent, strongly coupled protons (A1 = 0.45, A2 = 0.55, and A3 = 0.5 mT), identified by 2H substition. Incubation of oxidized formate dehydrogenase with cyanide resulted in an irreversible loss of enzyme activity, which could be restored by treatment with sulfide. Equimolar amounts of thiocyanate were released from cyanide-treated formate dehydrogenase, indicating the loss of one terminal sulfur ligand to molybdenum. Cyanide inactivation of the oxidized native enzyme resulted in a shift of the Mo(V) EPR signal to higher field (g1 = 2.005, g2 = 1.998, and g3 = 1.989) and a reduction in the complexity of the superhyperfine splitting pattern dur to loss of a strongly coupled, exchangeable proton, the remaining coupled proton yielding coupling constants of A1 = 1.05, A2 = -.85, and A3 = 0.88 mT, respectively. In addition, cyanide treatment altered the behavior of the Mo(V) EPR signal during potentiometric titration of the enzyme, yielding midpoint potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples of -319 and -321 mV, respectively. Comparison of the spectroscopic and thermodynamic properties of the native and cyanide-treated forms of formate dehydrogenase indicates the mode of inactivation to be analogous to that established for the related enzyme, xanthine, oxidase, resulting in the loss of a sulfur residue yielding the "desulfo" form of formate dehydrogenase.