Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia,the central process of biological nitrogen fixation.The mechanism of electron transfer from the P-cluster to the iron-molybdenum cofactor(FeMo-co)during the process of substrate reduction remains unclear.Based on our previous hypothesis,the dual pathway model,three mutants of Klebsiella oxytoca strain M5al(termed Nb102A,Ka431H and Ia423P)were constructed to map the H?/e-transfer pathway,in which b-102Asn,a-431Lys and a-423Ile of the molybdenum-iron(MoFe)protein of nitrogenases were replaced by alanine,histidine and proline,respectively.All three substitutions affected the diazotrophic growth,especially of the Ia423P strain with very low capability of diazotrophic growth and C2H2reduction activity.In a 42-L fermenter,the Ia423P strain was induced to the maximal C2H2reduction activity,which was only*17%of that of M5al.However,the transcription level of the nifD gene was approximately four-fold higher than that of the M5al strain,indicating that the low C2H2reduction activity of Ia423P was not because of downregulation of nifD expression.The maximal C2H2and H?reduction activities of the Ia423P MoFe protein were 13%and 21%of those of the wild-type MoFe protein,respectively.In conclusion,we propose that the changed properties of the Ia423P MoFe protein are related to the disruption of hydrogen bond formation between a-423Ile and homocitrate,and that a-423Ile serves as a gate to the Mo site in FeMo-co during the H?/e-transfer in nitrogenase.
Qingjuan GuoTao PengTianju ChangGang ZhangWei JiangYing LiJilun Li
