Ma K, Adams M W
Department of Biochemistry, University of Georgia, Athens 30602.
J Bacteriol. 1994 Nov;176(21):6509-17. doi: 10.1128/jb.176.21.6509-6517.1994.
Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 100 degrees C by the fermentation of carbohydrates yielding acetate, CO2, and H2 as the primary products. If elemental sulfur (S0) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction of polysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of 52,000 and 29,000 Da. SuDH contains flavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 95 degrees C of about 12 h and shows a 50% increase in activity after 12 h at 82 degrees C. The pure enzyme has a specific activity of 7 mumol of H2S produced.min-1.mg of protein-1 at 80 degrees C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 microM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (K(m) = 1.6 microM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (K(m) = 0.7 microM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of S(o) and polysulfide are discussed.
嗜热栖热菌是一种厌氧古菌，在100摄氏度时生长最佳，通过碳水化合物发酵产生乙酸盐、二氧化碳和氢气作为主要产物。如果向生长培养基中添加元素硫（S0）或多硫化物，也会产生硫化氢。嗜热栖热菌的细胞质氢化酶以铁氧化还原蛋白作为电子供体负责产生氢气，已证明它还能催化多硫化物还原为硫化氢（K. Ma、R. N. Schicho、R. M. Kelly和M. W. W. Adams，《美国国家科学院院刊》90:5341 - 5344，1993年）。我们现已从这种生物体的细胞质中纯化出一种酶，即硫化物脱氢酶（SuDH），它以NADPH作为电子供体催化多硫化物还原为硫化氢。SuDH是一种异二聚体，亚基分子量分别为52,000和29,000道尔顿。SuDH每摩尔含有黄素、约11个铁原子和6个酸不稳定硫化物原子，但未检测到其他金属。通过电子顺磁共振光谱对该酶进行分析表明存在四个铁硫中心，其中一个被NADPH特异性还原。SuDH在95摄氏度时的半衰期约为12小时，在82摄氏度下12小时后活性增加50%。纯酶在80摄氏度下以多硫化物（1.2 mM）和NADPH（0.4 mM）作为底物时，比活性为每分钟每毫克蛋白质产生7微摩尔硫化氢。表观Km值分别为1.25 mM和11 microM。NADH未被用作多硫化物还原的电子供体。嗜热栖热菌红素（K(m)=1.6 microM）也可作为SuDH的电子受体，并且SuDH以还原型嗜热栖热菌铁氧化还原蛋白（K(m)=0.7 microM）作为电子供体催化NADP的还原。文中讨论了SuDH的多种活性及其在S(o)和多硫化物代谢中的假定作用。
