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Temperature invariance of the nitrogenase electron transfer mechanism.

ABSTRACT: Earlier studies of electron transfer (ET) from the nitrogenase Fe protein to the MoFe protein concluded that the mechanism for ET changed during cooling from 25 to 5 °C, based on the observation that the rate constant for Fe protein to MoFe protein ET decreases strongly, with a nonlinear Arrhenius plot. They further indicated that the ET was reversible, with complete ET at ambient temperature but with an equilibrium constant near unity at 5 °C. These studies were conducted with buffers having a strong temperature coefficient. We have examined the temperature variation in the kinetics of oxidation of the Fe protein by the MoFe protein at a constant pH of 7.4 fixed by the buffer 3-(N-morpholino)propanesulfonic acid (MOPS), which has a very small temperature coefficient. Using MOPS, we also observe temperature-dependent ET rate constants, with nonlinear Arrhenius plots, but we find that ET is gated across the temperature range by a conformational change that involves the binding of numerous water molecules, consistent with an unchanging ET mechanism. Furthermore, there is no solvent kinetic isotope effect throughout the temperature range studied, again consistent with an unchanging mechanism. In addition, the nonlinear Arrhenius plots are explained by the change in heat capacity caused by the binding of waters in an invariant gating ET mechanism. Together, these observations contradict the idea of a change in ET mechanism with cooling. Finally, the extent of ET at constant pH does not change significantly with temperature, in contrast to the previously proposed change in ET equilibrium.

SUBMITTER: Mayweather D

PROVIDER: S-EPMC3487593 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Temperature invariance of the nitrogenase electron transfer mechanism.

Mayweather Diana D Danyal Karamatullah K Dean Dennis R DR Seefeldt Lance C LC Hoffman Brian M BM

Biochemistry 20121010 42

Earlier studies of electron transfer (ET) from the nitrogenase Fe protein to the MoFe protein concluded that the mechanism for ET changed during cooling from 25 to 5 °C, based on the observation that the rate constant for Fe protein to MoFe protein ET decreases strongly, with a nonlinear Arrhenius plot. They further indicated that the ET was reversible, with complete ET at ambient temperature but with an equilibrium constant near unity at 5 °C. These studies were conducted with buffers having a ...[more]

PMID: 23050654

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Project description:Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

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Temperature invariance of the nitrogenase electron transfer mechanism.

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Temperature invariance of the nitrogenase electron transfer mechanism.

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