Thorneley R N, Ashby G A
AFRC Institute of Plant Science Research, University of Sussex, Brighton, U.K.
Biochem J. 1989 Jul 1;261(1):181-7. doi: 10.1042/bj2610181.
The kinetics of oxidation of the Fe proteins of nitrogenases from Klebsiella pneumoniae (Kp2) and Azotobacter chroococcum (Ac2) by O2 and H2O2 have been studied by stopped-flow spectrophotometry at 23 degrees C, pH 7.4. With excess O2, one-electron oxidation of Kp2 and Ac2 and their 2 MgATP or 2 MgADP bound forms occurs with rate constants (k) in the range 5.3 x 10(3) M-1.S-1 to 1.6 x 10(5) M-1.S-1. A linear correlation between log k and the mid-point potentials (Em) of these protein species indicates that the higher rates of electron transfer from the Ac2 species are due to the differences in Em of the 4Fe-4S cluster. The reaction of Ac2(MgADP)2 with O2 is sufficiently rapid for it to contribute significantly to the high respiration rate of Azotobacter under N2-fixing conditions and may represent a new respiratory pathway. Excess O2 rapidly inactivates Ac2(MgADP)2 and Kp2(MgADP)2; however, when these protein species are in greater than 4-fold molar excess over the concentration of O2, 4 equivalents of protein are oxidized with no loss of activity. The kinetics of this reaction suggest that H2O2 is an intermediate in the reduction of O2 to 2 H2O by nitrogenase Fe proteins and imply a role for catalase or peroxidase in the mechanism of protection of nitrogenase from O2-induced inactivation.
在23摄氏度、pH 7.4条件下，利用停流分光光度法研究了肺炎克雷伯菌（Kp2）和褐球固氮菌（Ac2）固氮酶铁蛋白被O₂和H₂O₂氧化的动力学。在O₂过量的情况下，Kp2和Ac2及其与2 MgATP或2 MgADP结合的形式发生单电子氧化，速率常数（k）在5.3×10³ M⁻¹·s⁻¹至1.6×10⁵ M⁻¹·s⁻¹范围内。log k与这些蛋白质种类的中点电位（Em）之间的线性相关性表明，Ac2种类较高的电子转移速率是由于4Fe-4S簇的Em差异所致。Ac2（MgADP）₂与O₂的反应足够快，以至于在固氮条件下对褐球固氮菌的高呼吸速率有显著贡献，并且可能代表一种新的呼吸途径。过量的O₂会迅速使Ac2（MgADP）₂和Kp2（MgADP）₂失活；然而，当这些蛋白质种类的摩尔浓度比O₂浓度高4倍以上时，4当量的蛋白质被氧化且活性无损失。该反应的动力学表明，H₂O₂是固氮酶铁蛋白将O₂还原为2 H₂O过程中的中间体，这意味着过氧化氢酶或过氧化物酶在保护固氮酶免受O₂诱导失活的机制中起作用。
