THE HYDROGEN BINDING-SITE IN HYDROGENASE - 35-GHZ ENDOR AND XAS STUDIES OF THE NI-C ACTIVE FORM AND THE NI-L PHOTOPRODUCT
THE HYDROGEN BINDING-SITE IN HYDROGENASE - 35-GHZ ENDOR AND XAS STUDIES OF THE NI-C ACTIVE FORM AND THE NI-L PHOTOPRODUCT
复制标题
DOI：
10.1021/ja00066a032
复制
发表时间：
1993-06-30
影响因子：
15
通讯作者：
MARONEY, MJ
中科院分区：
文献类型：
--
作者：
WHITEHEAD, JP;GURBIEL, RJ;MARONEY, MJ
本文报道了用EPR、35-GHz ENDOR和X射线吸收（XAS）光谱法研究玫瑰果硫壳菌（Thiocapsa roseopersicina）氢化酶中H-2的结合。这些研究涉及对酶的活性形式进行的光谱，该活性形式显示指定为Ni-C的EPR信号。这种形式的酶是光敏的，并转化为光产物，表现出独特的EPR信号，Ni-L。H-1-ENDOR光谱揭示了与Ni-C中EPR活性中心强烈相互作用的两组质子。第一质子组（H1）是溶剂可交换的，源自二氢，并且具有类似于20 MHz的A（H1）值。第二质子组（H2）是不可溶剂交换的，具有类似于12 MHz的偶合常数A（H2），并且被分配给一个或多个半胱氨酸β-CH 2质子。在照射和EPR信号转化为Ni-L时，不再观察到可交换的H1质子集。在Ni-C的退火和再生后，再次观察到与H1质子集相关的特征。这是光处理和退火涉及类氢质子的解离和重组的直接证据。在准备使Ni-C和Ni-L最大化的样品上获得的XAS光谱没有揭示Ni位点之间的任何显著结构差异。
EPR, 35-GHz ENDOR, and X-ray absorption (XAS) spectroscopic studies of H-2 binding in hydrogenase from Thiocapsa roseopersicina are reported. These studies involve spectra taken on an active form of the enzyme that displays an EPR signal designated Ni-C. This form of the enzyme is light sensitive and is converted to a photoproduct that exhibits a unique EPR signal, Ni-L. H-1-ENDOR spectra reveal two sets of protons that interact strongly with the EPR-active center in Ni-C. The first proton set (H1) is solvent exchangeable, originates from dihydrogen, and has a value of A(H1) is similar to 20 MHz. The second proton set (H2) is not solvent exchangeable, has a coupling constant of A(H2) is similar to 12 MHz, and is assigned to one or more cysteine beta-CH2 protons. Upon irradiation and conversion of the EPR signal to Ni-L, the exchangeable H1 proton set is no longer observed. Upon annealing and regeneration of Ni-C, the feature associated with the H1 proton set is again observed. This is direct evidence that the photoprocess and annealing involve dissociation of a hydrogenic proton and recombination. XAS spectra taken on samples poised to maximize Ni-C and Ni-L do not reveal any significant structural difference between the Ni sites.