Soboh Basem, Linder Dietmar, Hedderich Reiner
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, Marburg, Germany.
Eur J Biochem. 2002 Nov;269(22):5712-21. doi: 10.1046/j.1432-1033.2002.03282.x.
From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe-4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different iron-sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.
从革兰氏阳性细菌嗜氢羧基热菌的膜组分中，纯化出一种催化CO转化为CO₂和H₂的酶复合物。该酶复合物在顶空含有5% CO时表现出最大的CO氧化：H₂释放酶活性（每毫克蛋白质450 U）。较高的CO浓度会抑制酶复合物中存在的氢化酶。为了达到最大活性，酶复合物必须用CO或强还原剂激活。该酶复合物还分别以每毫克蛋白质5900 U和180 U的活性催化甲基紫精的CO或H₂依赖性还原。发现该复合物由六个亲水性和两个疏水性多肽组成。确定了六个亲水性亚基的氨基末端序列，从而能够在嗜氢羧基热菌的初步基因组序列中鉴定编码基因。通过序列分析推断，该酶复合物由含镍一氧化碳脱氢酶（CooS）、含有四个[4Fe-4S]簇的电子传递蛋白（CooF）以及由四个亲水性亚基和两个膜整合亚基组成的膜结合[NiFe]氢化酶形成。复合物中的氢化酶部分与一小群[NiFe]氢化酶成员具有高度序列相似性，这些[NiFe]氢化酶与能量保守的NADH：醌氧化还原酶具有序列相似性。数据支持这样一种模型，即该酶复合物由两个催化位点组成，一个CO氧化位点和一个H₂形成位点，它们通过不同的含铁硫簇的电子传递亚基相连。该酶复合物在体内催化的放能氧化还原反应必须与能量守恒相偶联，最有可能是通过产生质子动力势来实现。
