PubMed · 5642386
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T Barker, C Eklund, O Wyss. 1968-03-27. Physical differences of virus-associated depolymerases.. https://doi.org/10.1016/0006-291x(68)90570-6
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The nitrogenase-catalyzed H(2) evolution and acetylene-reduction reactions using Ti(III) and dithionite (DT) as reductants were examined and compared under a variety of conditions. Ti(III) is known to make the all-ferrous Fe protein ([Fe(4)S(4)](0)) and lowers the amount of ATP hydrolyzed during nitrogenase catalysis by approximately 2-fold. Here we further investigate this behavior and present results consistent with the Fe protein in the [Fe(4)S(4)](0) redox state transferring two electrons ([Fe(4)S(4)](2+)/[Fe(4)S(4)](0)) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe(4)S(4)](2+)/[Fe(4)S(4)](1+)) using DT. MoFe protein specific activity was measured as a function of Fe:MoFe protein ratio for both a one- and a two-electron transfer reaction, and nearly identical curves were obtained. However, Fe protein specific activity curves as a function of MoFe:Fe protein ratio showed two distinct reactivity patterns. With DT as reductant, typical MoFe inhibition curves were obtained for operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple, but with Ti(III) as reductant the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple was functional and MoFe inhibition was not observed at high MoFe:Fe protein ratios. With Ti(III) as reductant, nitrogenase catalysis produced hyperbolic curves, yielding a V(max) for the Fe protein specific activity of about 3200 nmol of H(2) min(-1) mg(-1) Fe protein, significantly higher than for reactions conducted with DT as reductant. Lag phase experiments (Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702) were carried out at MoFe:Fe protein ratios of 100 and 300 using both DT and Ti(III). A lag phase was observed for DT but, with Ti(III) product formation, began immediately and remained linear for over 30 min. Activity measurements using Av-Cp heterologous crosses were examined using both DT and Ti(III) as reductants to compare the reactivity of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) and [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couples and both were inactive. The results are discussed in terms of the Fe protein transferring two electrons per MoFe protein encounter using the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with Ti(III) as reductant.
The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O(2) at the surface of diazotrophic prokaryotes protects nitrogenase from inactivation by O(2). Accordingly, it is assumed that, at increased ambient O(2) concentrations, nitrogenase activity depends on increased activities of a largely uncoupled respiratory electron transport system. The present review compiles evidence indicating that cellular O(2) consumption as well as both the activity and the formation of the respiratory system of Azotobacter vinelandii are controlled by the C/N ratio, that is to say the ratio at which the organism consumes the substrate (i.e. the source of carbon, reducing equivalents and ATP) per source of compound nitrogen. The maximal respiratory capacity which can be attained at increased C/N ratios, however, is controlled, within limits, by the ambient O(2) concentration. When growth becomes N-limited at increased C/N ratios, cells synthesize nitrogenase and fix N(2). Under these diazotrophic conditions, cellular O(2) consumption remains constant at a level controlled by the O(2) concentration. Control by O(2) has been studied on the basis of both whole cell respiration and defined segments of the respiratory electron transport chain. The results demonstrate that the effect of O(2) on the respiratory system is restricted to the lower range of O(2) concentrations up to about 70 microM. Nevertheless, azotobacters are able to grow diazotrophically at dissolved O(2) concentrations of up to about 230 microM indicating that respiratory protection is not warranted at increased ambient O(2) concentrations. This conclusion is supported and extended by a number of results largely excluding an obvious relationship between nitrogenase activity and the actual rate of cellular O(2) consumption. On the basis of theoretical calculations, it is assumed that the rate of O(2) diffusion into the cells is not significantly affected by respiration. All of these results lead to the conclusion that, in the protection of nitrogenase from O(2) damage, O(2) consumption at the cell surface is less effective than generally assumed. It is proposed that alternative factors like the supply of ATP and reducing equivalents are more important.
A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.