The protection of nitrogenase from oxygen in the cyanobacterium Anabaena sp. strain CA



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Protection of nitrogenase from the deleterious effects of oxygen was studied in the cyanobacterium, Anabaena sp. strain CA. Several types of experiments were performed in order to determine the effects of O₂ on nitrogenase. These included studies of the physiological response and adaptation of strain CA to various levels of O₂, the isolation and characterization of mutant strains of CA with increased sensitivity to O₂, and the analysis of protein composition and expression, especially within heterocysts, to increased O₂ tensions. Nitrogenase activity in CA was rapidly but reversibly inhibited by continuous exposure to 1% CO₂-99% O₂. Recovery was chloramphenicol sensitive. Nitrogenase-catalyzed H₂ evolution followed the same time course of inactivation and recovery as acetylene reduction. Evolution of H₂ was demonstrated under an atmosphere of 1% CO₂-99% O₂ following recovery. Other key physiological functions, including cell viability, photosynthetic oxygen evolution and the induction of heterocyst differentiation and nitrogenase synthesis were shown to be essentially unaffected by the presence or absence of O₂. Respiratory oxygen uptake increased significantly in diazotrophically-grown cultures compared to cultures grown on NH₄NO₃. The cells from these cultures also differed in their response to increased oxygen tensions. An oxygen-sensitive mutant showed high rates of both O₂-linked H₂ uptake and respiratory O₂ consumption. The enzymes of the nitrogenase complex in the mutant strain were destroyed by exposure to air levels of O₂. Upon exposure of cultures of CA to O₂, an alteration of the Fe-protein of nitrogenase was observed. This modification or alteration was completely reversible upon removal of O₂ from the cultures. The transition was not shown to be dependent on de novo protein synthesis. In Anabaena CA, the modification of the Fe-protein was shown to be specific for oxygen. It was concluded that while hydrogenase-linked and respiratory O₂ uptake systems may function in some limited capacity for the protection of nitrogenase, especially under low oxygen tensions, the primary protective and recovery mechanism at high oxygen tensions may involve the reversible alteration of the Fe-protein. Modification of the Fe-protein may increase the efficiency of electron transfer between components of nitrogenase under highly oxidized conditions or it may result in a type of conformational protection for the component proteins of the nitrogenase complex