Nitrogen Fixation in Bacteria and Higher Plants

Our knowledge of the biochemistry and biophysics of dinitrogen fixa­ tion has developed rapidly in the 15 years since the first N2-fixing enzyme system was successfully extracted from a bacterium. This peri­ od has produced a literature that now describes the N2 fixation reac­ tion and the nitrogenase enzyme itself in sophisticated terms, though a detailed reaction mechanism at the chemical level has not yet emerged. It is the purpose of the present monograph to present an in-depth re­ view, analysis, and integration of this research as is possible with a non-contributed publication and to relate this work to considera­ tions of N2 fixation that reach beyond the confines of the biochem­ ist's laboratory. The first section is directed as much toward the general science read­ er as toward the specialist. It covers the agricultural origins of man's interest in N2 fixation and also pertinent areas of taxonomy, physiology, and evolution. Ecological aspects of the subject include a comprehensive evaluation of the nitrogen cycle leading to a sub­ stantially greater estimate of the rate of global N2 fixation than previous ones. The treatment is of a survey fashion, in part to pro­ vide a general over-view of N2 fixation and in part to provide context for the biochemistry and biophysics that follow in the second section.

Contenu

I. The Diazotrophs.- 1 Recognition.- I. Historical Roots.- A. The Agricultural Imperative.- B. Early Experimentation.- II. Detection of Diazotrophs and Nitrogenase.- A. 15N2 Method.- B. Ammonia and Manometric Methods.- C. Acetylene Reduction Method.- D. Other Methods.- 2 Description and Classification of Diazotrophs.- I. Free-Living Diazotrophs.- A. Taxonomy and Characteristics.- 1. Bacteria.- 2. Blue-Green Algae.- B. Associative Symbioses.- 1. Loose Associations.- a) Interactions Among Microorganisms.- b) Phylloplane.- c) Rhizosphere.- 2. Associations Involving Morphological Modification or Accommodation.- 3. Associations with Animals.- II. Obligatory Symbioses.- A. Non-Legume Angiosperms.- B. Legumes.- 1. Rhizobia.- 2. Establishment of an Effective Legume Symbiosis.- 3. Tissue Culture Symbiosis.- Concluding Remarks.- 3 Perspectives.- I. Evolution.- A. N2 Fixation in Free-Living Organisms.- B. N2 Fixation in Symbioses.- II. Role of Diazotrophs in Global Nitrogen Economy.- A. Nitrogen Cycle.- 1. Nitrogen Sinks.- 2. Nitrogen Transformations and Transfers.- a) Soil-Water Pool.- b) Atmosphere.- Concluding Remarks.- B. Amount of N2 Fixed.- 1. N2 Fixation Rates in Various Ecosystems.- a) Cultivated Land.- b) Forests and Grasslands.- c) Unused Land.- d) Marine.- 2. Comparison with Previous Estimates.- II. Biochemistry and Biophysics.- 4 An Equation for the Nitrogenase Reaction.- I. The Clostridium - Pyruvate N2-Fixing System.- II. ATP.- III. Azotobacter Nitrogenase and Dithionite.- IV. H2 Evolution and ATP Hydrolysis: Nitrogenase Activity without N2.- V. Alternate Substrates for Nitrogenase.- VI. H2, N2, Hydrogenase and Nitrogenase - A Special Relationship?.- 5 Nitrogenase: The Catalyst.- I. Procurement.- A. Factors Affecting Nitrogenase Biosynthesis.- 1. Genetic Basis.- 2. Culture Conditions.- a) Metabolic Control.- b) General Considerations.- B. Extraction and Purification of Nitrogenase.- II. Properties of Mo-Fe Protein and Fe Protein.- A. Nitrogenase Proteins of Various Diazotrophs.- 1. Azotobacter vinelandii.- a) Mo-Fe Protein.- b) Fe Protein.- 2. Clostridium pasteurianum.- a) Mo-Fe Protein.- b) Fe Protein.- 3. Kiebsiella pneumoniae.- a) Mo-Fe Protein.- b) Fe Protein.- 4. Azotobacter chroococcum.- 5. Mycobacterium flavum.- 6. Chloropseudomonas ethylicum.- 7. Soybean Bacteroids.- B. Summary of Characteristics of the Nitrogenase Proteins.- 1. Mo-Fe Protein.- 2. Fe Protein.- III. Properties of Nitrogenase.- A. The Question of Nitrogenase Composition.- B. Physical-Chemical Characteristics of Nitrogenase.- C. Stability of Nitrogenase.- 1. O2 Sensitivity.- 2. Cold Lability.- D. Integrity of Reconstituted Nitrogenases.- 6 Nitrogenase: The Catalysis.- I. Nitrogenase Turnover.- A. Enzyme Concentration and Composition.- B. ATP.- 1. ATP and Reaction Rate.- a) Relationship of ATP, Enzyme Concentration and Activity.- b) Michaelis Constant.- c) ADP and Salt Inhibition.- 2. The ATP:2-Electron Ratio.- 3. Reductant-Independent ATP Hydrolysis.- 4. Other ATP Effects.- C. Dithionite.- D. External Factors.- 1. pH.- 2. Temperature.- 3. Inhibitors.- II. Substrate Reduction.- A. Electron Allocation: H3O+vs. Exogenous Reducible Substrate.- B. Specificity and Inhibition.- C. The Reducible Substrates.- 1. N2.- 2. Azide.- 3. N2O.- 4. Nitriles.- a) Cyanide.- b) Alkyl Cyanides.- c) Alkenyl Cyanides.- 5. Isonitriles.- 6. Alkynes.- Concluding Remarks on Substrate Reduction.- 7 Mechanism.- I. A Model for the Formation of Catalytically Active Species.- A. Protein and Ligand Complexes of the Model.- 1. Fe Protein · ATP · Mg Complex.- 2. Mo-Fe · Protein · Fe Protein Complexes.- B. Reactivity of the Model.- 1. Dilution Effect.- a) The Need for 2 Fe Proteins.- b) Effect of ATP Concentration.- c) ADP and Salt Effects.- 2. ATP Hydrolysis.- 3. Nature of ATP Saturation Curves and Reciprocal Plots for Km (ATP) Evaluation.- 4. Reaction of ATP with the Mo-Fe Protein · Fe Protein Complexes.- C. Allosteric Implications.- D. The Mechanistic Enigmas of ATP Hydrolysis and Electron Transfer.- II. Reducible Substrate Complexation and Reduction.- A. Locus of Active Site.- B. Role of Metals and Implications of Abiological Systems.- 1. Nitriding Systems.- 2. N2 Complexes in Aprotic Media.- 3. Homogeneous Protonic Catalysis.- C. Active Site Model and Reduction Sequence.- 8 Cellular Accommodation of Nitrogenase.- I. Localization of Nitrogenase.- II. Electron-Donating Systems.- A. Clostridium pasteurianum.- B. Azotobacter vinelandii.- C. Azotobacter chroococcum.- D. Mycobacterium flavum.- E. Bacillus polymyxa.- F. Chromatium.- G. Chloropseudomonas ethylicum.- H. Anabaena cylindrica.- I. Soybean Nodule Bacteroids.- III. Ammonia Assimilation.- IV. Other Enzymes and Proteins Associated with N2 Fixation.- A. Hydrogenase.- B. Leghemoglobin.- C. Other Proteins.- 1. Cytochromes.- 2. Nitrate Reductase.- References.