Schneider K, Müller A, Krahn E, Hagen W R, Wassink H, Knüttel K H
Fakultät für Chemie, Lehrstuhl für Anorganische Chemie I, Universität Bielefeld, Germany.
Eur J Biochem. 1995 Jun 1;230(2):666-75. doi: 10.1111/j.1432-1033.1995.0666h.x.
In the presence of molybdate (1 microM) 2-3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm). The maximal specific activity obtained was 145 nmol ethylene reduced . min-1 . mg protein-1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo-independent nitrogenase does not exist. However, the molybdate-containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases. The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH-dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.
在含有钼酸盐（1微摩尔）、2 - 3.5%氧气且以蔗糖作为碳源的条件下，嗜微氧固氮氢氧化细菌自养黄色杆菌GZ29以固氮方式生长，最短倍增时间为2.5小时，计算得出的吸光度（546纳米）最高可达52。获得的最大比活性为145纳摩尔乙烯还原量·分钟⁻¹·毫克蛋白⁻¹（粗提取物）。以蛋氨酸作为氮源时，钼固氮酶的阻遏解除至相当水平。钒化合物既不刺激生长也不刺激固氮酶活性。不添加钼酸盐时，固氮生长和固氮酶活性降至极低水平。负责钼酸盐饥饿细胞中残余活性的固氮酶含有钼酸盐但不含其他杂金属原子。这些结果表明，在自养黄色杆菌中不存在不依赖钼的固氮酶。然而，含钼酸盐的固氮酶表现出一些类似于替代固氮酶的特性。钼铁蛋白（组分1，Xa1）与两个此前未检测到的小分子多肽分子（摩尔质量13.6 kDa）共纯化，并且不仅能将乙炔还原为乙烯，还能部分还原为乙烷。在特定条件下，即在碱性pH值的Tris/HCl缓冲液中，以柠檬酸钛（III）作为电子供体，在高组分1/组分2比例以及低的、不饱和乙炔浓度下，可检测到高达5.5%的乙烷。与乙烷相对产率的pH依赖性增加并行的是，总活性（乙炔和氮气还原速率）降低，并且S = 3/2铁钼辅因子的ESR信号分裂为三个具有不同菱形度的信号[E/D值分别为0.036（信号I）、0.072（信号II）和0.11（信号III）]。pH值越高，两个新的铁钼辅因子信号的强度越明显。使用柠檬酸钛（III）代替连二亚硫酸钠作为还原剂可进一步增强这些信号。
