Khangulov S V, Gladyshev V N, Dismukes G C, Stadtman T C
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Mar 10;37(10):3518-28. doi: 10.1021/bi972177k.
Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.
来自大肠杆菌的甲酸脱氢酶H（FDH(Se)）在其多肽中含有一个钼蝶呤鸟嘌呤二核苷酸辅因子和一个硒代半胱氨酸残基。由FDH(Se)催化的在富含18O的水中13C标记的甲酸氧化产生不含18O标记的13CO2气体，这表明该酶不属于将水中的氧掺入产物的含钼蝶呤氧转移酶大类。活性位点的一个不寻常的钼中心在还原的Mo(IV)状态下以四方锥几何构型与来自一对蝶呤辅因子的四个赤道二硫烯硫原子以及硒代半胱氨酸-140残基的一个硒原子配位[博因顿，J.C.，格拉迪谢夫，V.N.，坎古洛夫，S.V.，斯塔特曼，T.C.，和孙，P.D.（1997年）《科学》275卷，1305 - 1308页]。Mo(V)状态的电子顺磁共振光谱表明其几何构型类似于Mo(IV)中心。最强的配体场分量可能是单个轴向硒原子，产生基态轨道构型Mo(dxy)。基于标量和偶极超精细分量与原子77Se的比较，估计Mo - Se键的共价程度为未成对电子自旋密度的17 - 27%，该自旋密度存在于价4s和4p硒轨道中。Mo(VI)状态将甲酸进行两电子氧化，使Mo转变为还原的Mo(IV)状态，同时甲酸盐质子Hf+转移到附近的碱基Y - 。向Fe4S4中心转移一个电子将Mo(IV)转变为可通过电子顺磁共振检测到的Mo(V)状态。Y - 位于与[Mo - Se]中心的磁接触范围内，这由其强烈的偶极1Hf超精细耦合表明。甲酸诱导的Mo(V)状态的光解消除了来自YHf的1Hf超精细分裂，表明该基团的光异构化或质子向更远的质子受体基团A - 的光转移。光解对与[77Se]硒代半胱氨酸的77Se - 超精细相互作用的微小影响表明Y - 基团不是硒原子，而是可能是位于靠近[Mo - Se]中心的假定底物结合口袋中的His141残基的咪唑环。我们提出，Hf+从甲酸转移到活性位点碱基Y - 在热力学上与甲酸分子的两电子氧化耦合，从而促进CO2的形成。在正常生理条件下，当电子流不受电子终端受体限制时，Fe4S4将Mo(IV)中心氧化时释放的能量用于YHf的去质子化以及Hf+逆热力学势的转移。
