menaquinone-6 and formic-acid

In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK(6)) or 8-methylmenaquinone (MMK(6)) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK(6) from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

Topics: Anaerobiosis; Bacterial Proteins; Cell-Free System; Electron Transport; Formate Dehydrogenases; Formates; Hydrogen; Klebsiella pneumoniae; Membranes, Artificial; Nitrogen; Oxygen; Transcription Factors; Vitamin K 2; Wolinella

Topics: Binding Sites; Catalysis; Catalytic Domain; Cell Membrane; Crystallography, X-Ray; Electron Transport; Escherichia coli; Formate Dehydrogenases; Formates; Guanine Nucleotides; Hydrogen Bonding; Iron-Sulfur Proteins; Membrane Potentials; Nitrates; Organometallic Compounds; Oxidation-Reduction; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Proton-Motive Force; Protons; Vitamin K 2

The structure of the membrane protein formate dehydrogenase-N (Fdn-N), a major component of Escherichia coli nitrate respiration, has been determined at 1.6 angstroms. The structure demonstrates 11 redox centers, including molybdopterin-guanine dinucleotides, five [4Fe-4S] clusters, two heme b groups, and a menaquinone analog. These redox centers are aligned in a single chain, which extends almost 90 angstroms through the enzyme. The menaquinone reduction site associated with a possible proton pathway was also characterized. This structure provides critical insights into the proton motive force generation by redox loop, a common mechanism among a wide range of respiratory enzymes.

Topics: Binding Sites; Catalysis; Catalytic Domain; Cell Membrane; Crystallography, X-Ray; Electron Transport; Escherichia coli; Formate Dehydrogenases; Formates; Guanine Nucleotides; Hydrogen Bonding; Iron-Sulfur Proteins; Membrane Potentials; Models, Molecular; Nitrate Reductases; Oxidation-Reduction; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Proton-Motive Force; Protons; Pterins; Vitamin K 2