Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by phosphorus-31 NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation

Abstract

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.

Reconciling the incompatible: N2 fixation And O2

“…Nevertheless, nitrogenase may make a significant contribution to O^ consumption in A. chroococcum. Whilst not suggesting that uptake of O^ by nitrogenase can account for the entire capacity of A. chroococcum to consume O,, Thorneley & Ashby (1989) speculate that the increase in respiration seen on transfer from O,,-limited to Og-sufficient conditions could be explained in this way. Moreover, a significant role for nitrogenase-catalyzed O, consumption may resolve some of the difficulties, referred to above, in understanding the interrelations between respiratory Og uptake and N2 fixation in Azotobacter.…”

“…On the other hand, it has recently been shown that the fiavodoxin that acts as an electron donor to the nitrogenase of K. pneumoniae (the nifF product) is subject to covalent modification. Attachment of a coenzyme A residue causes inactivation of this fiavodoxin and may constitute part of the regulatory mechanism associated with reversible loss of nitrogenase activity during transient exposure to Og (Thorneley et aL, 1992). However, there is, as yet, no direct evidence to support this suggestion.…”

“…Nitrogenase can account for as much as 40 % of the cellular protein of diazotrophs (Jouanneau, Wang & Vignais, 1985). Thorneley & Ashby (1989), taking a less extreme value of 10%, calculated that the concentration of nitrogenase relative to intracellular Og would be such that the enzyme in Azotobacter chroococcum would reduce O, to HgO, without undergoing any O^-induced inactivation. However, catalase and/or peroxidases would be needed to remove the H^O, produced by this reaction.…”

Immunochemical detection of CoA-modified mitochondrial matrix proteins

“…Samples : (A) 100 µl of acid-soluble supernatant (total volume 330 µl) ; (B) for reference, 3.8 nmol of adenosine 3h :5h diphosphate (P-AMP), 872 nmol of dithioerythritol (DTE), 1.9 nmol of AMP, 1.1 nmol of CoA (CoASH) and 0.5 nmol of oxidized CoA (CoASSCoA) were applied to (6) groups in comparison with the unmodified enzyme [22]. Covalent binding of CoA through a disulphide bond has also been demonstrated for the mutant β-subunit of F " -ATPase (where a Tyr residue has been replaced by Cys) [23] and for a flavodoxin of Klebsiella pneumoniae [24].…”

Roulette mutagenesis of the FMN‐binding site of Klebsiella pneumoniae flavodoxin

“…Tertiary structures have also been determined crystallographically for flavodoxins from Clostridiurn beijerinckii MP, Desulfovibrio vulgaris and Chondrus crispus (see [2] and references therein) and by two-dimensional 'H-NMR Tor the Mega;,-phaera elsdenii flavodoxin [31. The crystal structure of the Azotobacter chrnococcum flavodoxin, an electron donor to nitrogenase showing strong sequence similarity to the K. pneunzoniae moleculc, has recently been completed [4]. We have investigated the contribution to structure and function of three residues in the K. pneurnoniae flavodoxin, Gly12, Lysl3 and Thrl4, which form part of the binding cleft for the prosthetic group, FMN, and are close in the tertiary structure to Cys68, which we have recently shown to be covalently but reversibly modified by coenzyme A [ 5 ] . In known structures, the strongly conserved residue Glyl2 occupies the fourth position in the reverse turn between the first p strand and the first u: helix.…”