Maness Pin-Ching, Smolinski Sharon, Dillon Anne C, Heben Michael J, Weaver Paul F
The National Renewable Energy Laboratory, Golden, Colorado 80401-3393, USA.
Appl Environ Microbiol. 2002 Jun;68(6):2633-6. doi: 10.1128/AEM.68.6.2633-2636.2002.
A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.
与嗜胶红游动菌中一氧化碳氧化途径相关的氢化酶表现出对氧气的耐受性。当全细胞或无膜部分纯化的氢化酶在全空气（21% O₂，79% N₂）中搅拌时，通过使用还原甲基紫精的厌氧测定法确定，其氢气释放活性的半衰期分别为20小时或6小时。当部分纯化的氢化酶在含有3.3%或13% O₂的气氛中搅拌15分钟并通过氢 - 氘（H - D）交换测定法评估时，其同位素交换率分别保留了近80%或60%。当该酶悬浮液随后返回厌氧气氛时，超过90%的H - D交换活性得以恢复，这反映了该氢化酶对氧气失活的可逆性。与大多数氢化酶一样，与一氧化碳相关的氢化酶对一氧化碳极为敏感，在溶解的一氧化碳浓度为3.9微摩尔时，50%的活性受到抑制。当原核来源的铁氧化还原蛋白作为直接电子介质时，只要它们被含有活性水分解活性的菠菜类囊体膜光还原，就能检测到与一氧化碳相关的氢化酶产生氢气。基于其对氧气的显著耐受性，讨论了这种氢化酶的潜在应用。
