W. E. Newton

New Horizons in Nitrogen Fixation

this volume) to become associated with apodinitrogenase. The role of general chaperonins in promoting accumulation of nif gene products (Govezensky et al.1991) will be discussed in chapters of this section and may suggest roles for some of the nif-specific gene products to which no role has yet been ascribed. It is hoped that by the time of the next meeting, the enzymatic or transfer reactions carried out by the various nif genes involved in metal cluster assembly will be known in detail. Acknowledgements. The author wishes to acknowledge the contributions of his collaborators, GP Roberts, VK Shah, RM Allen, MJ Homer, R Chatterjee, J Allen and J Roll at the University of WI. Work from the laboratories of the author and his collaborators has been generously supported by the NIH and NRI/CGP.

Advances in Nitrogen Fixation Research

Cultured isolates of Anabaena azollae provide a suitable tool for studying the metabolism, transport, cell composition and antigenic properties of the cells. The isolates of Anabaena azollae from Azolla caroliniana and Azolla filiculoides exhibit antigenic homology in the quantitative immunoassays employed, suggesting that they may stem from the same parental Anabaena. These isolates were also similar in respect to the fructose supported N2-fixation activity: the fructose is taken up actively by the cells of Anabaena azollae and although it does not support the growth of Anabaena, it carries a series of changes involving cell composition, differentiation and metabolism. Respiration was facilitated, the amount of storage products increased, and the frequency of heterocysts was increased. Sucrose and glucose stimulated respiration, but showed limited effect on N2-fixation. The uptake of fructose, dependent on ATP synthesis, is recovered faster in starved cells than N2-fixation activity. Fructose carrier seems to be constitutive in the cultured isolate of Anabaena azollae grown autotrophically. The lectins identified in Anabaena azollae Newton cells are constitutive as well, and their actual role in the symbiosis with Azolla has not been verified yet. They may be involved in the regulation and control of the development of Anabaena in the leaf cavi ty and, poss ibly, trigger the exchange of me taboli tes between the hos t Azolla and the N2-fixing Anabaena.

Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster.

DNA fragments coding for the structural genes for Azotobacter vinelandii nitrogenase have been isolated and sequenced. These genes, nifH, nifD and nifK, code for the iron (Fe) protein and the alpha and beta subunits of the molybdenum-iron (MoFe) protein, respectively. They are arranged in the order: promoter:nifH:nifD:nifK. There are 129 nucleotides separating nifH and nifD and 101 nucleotides separating nifD and nifK. The amino acid (aa) sequences deduced from the nucleotide sequences are discussed in relation to the prosthetic group-binding regions of the nifHDK-encoded polypeptides.

A systematic x-ray absorption study of molybdenum complexes. The accuracy of structural information from extended x-ray absorption fine structure

X-ray absorption spectra have been collected using synchrotron radiation for a number of mononuclear and dinuclear molybdenum complexes containing carbon, nitrogen, oxygen, and sulfur donor atoms. The extended fine structure (EXAFS) of the Mo absorption edge has been analyzed by a method which combines Fourier transform and curve-fitting techniques. Parameterized phase shift and amplitude functions for describing Mo-C, Mo-N, Mo-0, Mo-S, and Mo-Mo interactions were obtained from spectra of Mo(CO)6, M o ( N C S ) ~ ~ , MOO^^-, M o ( S ~ C ~ H ~ ) ~ , and M0204cys2~-. Application of these phase shifts and amplitudes to the EXAFS of other compounds yielded distance determinations to an accuracy consistently better than 0.03 A for atoms bound to Mo. The number and type of coordinating atoms were also determined with a reasonable degree of certainty. This work demonstrates the applicability (and limitations) of EXAFS for providing structural information about a specific absorbing center under noncrystalline conditions, and it lays a foundation for the analysis of the x-ray absorption spectra of nitrogenase and other Mo proteins.

Genomes and genomics of nitrogen-fixing organisms

Preface to the Series. Preface. List of Contributors. 1: Origins of Genomics in Nitrogen-Fixation Research G. Davila and R. Palacios 1. Introduction 2. Symbiotic Organisms 3. Free-Living Organism 4. Conclusion References 2: Genomics of Diazotrophic Archaea J.A. Leigh 1. Introduction 2. The Core nif-gene Cluster 3. Other nif Genes 4. Other Nitrogen Assimilatory Genes 5. PII Proteins References 3: Genomic Aspects of Nitrogen Fixation in the Clostridia J.-S. Chen 1. Introduction 2. The Nitrogen-Fixing Clostridia 3. The Genome of the Clostridia 4. Organization of the Nitrogen-Fixation Gene Cluster 5. Regulatory Genes for Nitrogen Metabolism 6. Altenative Nitrogen-Fixation (anf) Genes 7. Genes for Nitrogen Assimilation 8. Concluding Remarks References 4: The Genome of the Filamentous Cyanobacterium Nostoc punctiforme J.C. Meeks 1. Introduction 2. Phenotypic Traits of N. punctiforme 3. Overview of the N. punctiforme Genome 4. N. punctiforme genes Involved in Heterocyst Formation, Nitrogenase Expression, and Ammonia and Nitrate Assimilation 5. Summary and Conclusions Acknowledgements References 5: The nif Genes of Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas palustris R. Haselkorn and V. Kapatral 1. Introduction 2. Regulation of the Nitrogen-Fixation System 3. Operon Structure and Gene Organization Acknowledgement References 6: Genomic Architecture of the Multiple Replicons of the Promiscuous Rhizobium Species NGR234 P. Mavingui, X. Perret and W. J. Broughton 1. Introduction 2. Promiscuity of NGR234 3. Structural Organization of the NGR234 Genome 4. Coding Capacity of eth NGR234 Genome 5. Conclusions and Perspectives References 7: Facets of the Bradyrhizobium japonicum 110 Genome M. Gottfert, H. Hennecke and S. Tabata 1. Introduction 2. Materials and Methods 3. Genome Characteristics 4. Perspectives References 8: pSymA of Sinorhizobium meliloti: Nitrogen Fixation and More M.J. Barnett and M.L. Kahn 1. Introduction 2. History 3. The pSymA Sequence Project 4. General Features of pSymA 5. Comparative Genomics 6. Plasmid Biology 7. Elements of External Origin 8. Transfer RNA Genes 9. Nodulation Genes 10. Nitrogen-Fixation Genes 11. Carbon and Nitrogen Metabolism 12. Chemotaxis and Pilus Formation 13. Transport 14. Regulation and Signal Transduction 15. Stress Responses 16. Sulfur Metabolism 17. Orphan Genes 18. Genome-Wide Analysis 19. A Strategy for Analyzing pSymA of S. meliloti References 9: Rhizobium etli Genome Biology G. Davila, V. Gonzalez, M.A. Ramirez-Romero and O. Rodriguez 1. Introduction 2. Rhizobium etli Genome Structure 3. Rhizobium Genome Plasticity 4. Rhizobium etli Taxomony and Evolution Acknowledgements References 10: The Dawn of Functional Genomics in Nitrogen-Fixation Research S. Encarnacion 1. Introduction 2. Functional Genomics - The Role of Gene-Expression Studies 3. The Transcriptome 4. Transcriptomics in Nitrogen-Fixation Research 5. Transcriptomics in Plants during Symbiotic Nitrogen Fixation 6. The Proteome 7. Proteomics and Nitrogen-Fixation Research 8. Proteomics in Plants during Symbiotic Nitrogen Fixation 9. Proteomics in Concert with Transcriptomics 10. Global Approaches to Study the R. etli-P. vulgaris Interaction 11. Protein-Protein Interactions: Applications of Molecular Maps 12. Transcriptomics, Proteomics and Bioinformatics 13. Conclusions Acknowledgements References 11: Transcriptomics in Sinorhizobium meliloti A. Becker and F. J. De Bruijn 1. Introduction to Transcriptomics 2. Introduction to the Biological System 3. Sinorhizobium meliloti Microarray 4. S. meliloti Macroarrays 5. Conclusions and Perspective Acknowledgements References 12: Genome Dynamics in Rhizobial Organisms R. Palacios and M. Flores 1. Introduction 2. Reiterated Sequences 3. Genomic Instability 4. Natural Gene Amplification 5. Artificial Gene Amplification 6. Dynamics of Genome Architecture 7. Prediction of Genome Rearrangements 8. Identification of Genome Rearrangements 9. Artificial Selection of Genomic Rearrangements 10. Natural Genomic Design 11. Concluding Remarks Acknowledgements References 13: Impact of Genomics on the Reconstruction of Evolutionary Relationships of Nitrogen-Fixing Bacteria and Implications for Taxonomy P. Van Berkum and B. D. Eardly 1. Systematics 2. Current Reflections for Evolution of Diazotrophy 3. Reconstruction of Evolutionary Relationships among Members of the Kingdom Monera 4. The Rapid Spread of Antibiotic Resistance: Implications of Reticulate Microbial Evolution 5. Mechanisms of Horizontal Gene Transfer in Microbes 6. Significance of Horizontal Gene transfer in Nature 7. Evidence for Lateral Gene Transfers and Recombination in Microbial Genomes 8. Genomic Islands 9. Microbial Evolution and Genetic Recombination 10. Established Species Concepts Applied to Bacteria 11. A Proposed Unified Species for Bacteria 12. Relevant Insights from Recent Genomic Comparisons 13. Implications and Future Strategies AcknowledgementsReferences 14: The Phylogeny and Evolution of Nitrogenases J.P.W. Young 1. Introduction 2. The Genetic Organization of Nitrogenase Genes 3. Nitrogenase Genes from Genome Sequencing Projects 4. Organization of the Nitrogenase Genes 5. Evolutionary Relationships of the Nitrogenase Genes 6. Nitrogenase Phylogeny versus Organism Phylogeny 7. Nitrogenase Genes in their Genomic Context 8. Conclusions and Prospects References Subject Index

Evidence that conserved residues Cys‐62 and Cys‐154 within the Azotobacter vinelandii nitrogenase MoFe protein α‐subunit are essential for nitrogenase activity but conserved residues His‐83 and Cys‐88 are not

Metallocluster extrusion requirements, interspecies MoFe‐protein primary sequence comparisons and comparison of the primary sequences of the MoFe‐protein subunits with each other have been used to assign potential P‐cluster (Fe‐S cluster) domains within the MoFe protein. In each β unit of the MoFe protein, subunit domains, which include potential Fe‐S cluster ligands Cys‐62, His‐83, Cys‐88 and Cys‐154, and β‐subunit domains, which include potential Fe‐S cluster ligands Cys‐70, His‐90, Cys‐95 and Cys‐153, are proposed to comprise nearly equivalent P‐cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the α‐ subunit Cys‐62, His‐83, Cys‐88 and Cys‐154 positions by site‐directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates. MoFe—protein acetylene‐reduction activities, and whole‐cell S 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe‐protein α‐submit residues, Cys‐62 and Cys‐154, are probably essential for MoFe‐protein activity but that His‐83 and Cys‐88 residues are not. These results indicate either that His‐83 and Cys‐88 do not provide essential P‐cluster ligand or that a new cluster‐ligand arrangement is formed in their absence.

Structural Characterization of CO-Inhibited Mo-Nitrogenase by Combined Application of Nuclear Resonance Vibrational Spectroscopy, Extended X-ray Absorption Fine Structure, and Density Functional Theory: New Insights into the Effects of CO Binding and the Role of the Interstitial Atom

The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm–1 mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm–1, additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by 13CO isotope shifts). The EXAFS for wild-type N2ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal −CO and a partially reduced −CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational “shake” modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N2 reactivity of N2ase are discussed.

Iron-molybdenum cofactor of Axotobacter vinelandii nitrogenase: oxidation-reduction properties and structural insights

Abstract Structural, oxidation-reduction and compositional probes of the iron-molybdenum cofactor (FeMoco) from the MoFe protein of Azotobacter vinelandii nitrogenase have been undertaken, via variety of physicochemical techniques, to gain insight into the nature of this entity. Using highly concentrated (> 1 mM) solutions of extruded FeMoco in N-methylformamide (NMF), details of its oxidation-reduction behavior have been garnered for the first time. A combination of cyclic voltammetry, potentiometry and electron paramagnetic resonance (EPR) spectroscopy has shown that, of the two “quasi-reversible”, couples observed, the more-positive couple (at ca −0.3 V vs NHE) corresponds to the “oxidized”-to-“semi-reduced” change as demonstrated by the production of the unique S = 3 2 EPR signal of FeMoco on titration through this couple with aliquots of aqueous dithionite. Plots of EPR intensity vs added electrons and Nernst plots of these data are consistent with the involvement of a single electron in this process. The more negative couple at ca −1.0 V may well involve the substrate-reducing state of FeMoco. No synthetic MoFeSO compound has yet been found which duplicates these electrochemical properties. X-ray absorption spectroscopy studies at the Mo K-edge of similar highly concentrated FeMoco solutions have produced high-quality data in terms of the near-edge (XANES) spectra. Comparisons with similar data sets from FeMoco bound within the protein matrix indicate some (but not radical) structural flexibility or variability around the Mo site. The XANES of a variety of synthetic Mo compounds, which fall into three major categories: (i) Mo in an approximately tetrahedral environment, e.g. [Fe(MoS4)2]3−, which exhibits two inflection points on the Mo absorption edge at ca 20,004 and 20,010 eV; (ii) Mo in all-sulfur, pseudo-octahedral environment, e.g. the dicubane [Mo2Fe6S8(SEt)9]3−, which has a single inflection point at ca 20,010 eV; and (iii) Mo in a mixed sulfur-oxygen, pseudo-octahedral environment, e.g. [Mo2Fe6S8(Set)6(OMe)3]3−, which has two inflection points at ca 20,012 and 20,020 eV, have also been obtained. The XANES of FeMoco, both within and outside the protein, show a marked similarity to those of compounds of the third category, indicating Mo nearest neighbors of 3O (or N) and 3S from anionic ligands. These results are consistent with the structural data derived from the deconvolution of the EXAFS of similar extruded and protein-bound FeMoco samples. Oxidative decomposition studies of isolated FeMoco are also consistent with a coordination environment of only three S atoms around Mo.

Another role for CO with nitrogenase? CO stimulates hydrogen evolution catalyzed by variant Azotobacter vinelandii Mo-nitrogenases.

A likely entry/exit path for nitrogenase substrates, products, and/or protons involves residues α277(Arg), α192(Ser), and α356(Gly), all of which are highly conserved among MoFe proteins from different organisms. The α192(Ser) and α277(Arg) residues form part of a hydrogen-bonded network that also involves α195(His), which interacts with a FeMo cofactor-based sulfide. The terminal amino groups of α277(Arg) are also hydrogen-bonded directly to α281(Tyr), which resides at the surface of the MoFe protein. Individual amino acid substitutions placed at position α277 or α192 resulted in a variety of effects on the catalytic and/or spectroscopic properties of the resulting variant MoFe protein. Of particular interest was the effect of CO on H2 evolution catalyzed by three MoFe protein variants, α277(Cys), α192(Asp), and α192(Glu). All three variants exhibited CO stimulation of H2 evolution under high-electron flux conditions but not under low-electron flux conditions. This observation is best explained by these variants being redox-compromised but only at the most reduced redox states of the MoFe protein. Normally, these states are accessed and operational only under high-electron flux conditions, and the effect of added CO is to prevent access to these most reduced redox states, resulting in a normal rate of catalysis. Furthermore, via correlation of the effect of pH changes on H2 evolution activity for both the wild type and the α277(Cys) MoFe protein variant under argon, with or without 10% CO present, likely pathways for the delivery of a proton to the FeMo cofactor were identified.

Synthesis and chemistry of some binuclear oxomolybdenum(V) xanthate (O-alkyl dithiocarbonate) complexes

A simple method for the preparation of pure µ-oxo-bis[bis(O-alkyl dithiocarbonato)oxomolybdenum(V)] complexes, [Mo2O3(RO·CS·S)4](I; R = Me, Et, Pri, Bun, or Bui), and difficulties encountered with previous preparative methods are reported. Assignments of molybdenum–oxygen stretching frequencies in the i.r. spectra have been made and visible spectra have been reinvestigated. Reaction with dialkylamines, alcohols, or hydrogen sulphide results in loss of xanthate ligand and produces, in the last two cases, a series of new di-µ-sulphido-bis[(O-alkyl dithiocarbonato)oxomolybdenum(V)] complexes, [Mo2O2S2(RO·CS·S)2](II). Spectroscopic studies of these products indicate that those containing unbranched O-alkyl dithiocarbonate groups are polymers in the solid state, which dissociate in solution. Complexes (I) and (II) react with excess of xanthate ligand to produce the ion [Mo2O2S2(O·CS·S)2]2–, (III).