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.

A method of randomising specific regions of coding sequences has been devised which utilises the Lac phenotype to identify mutants. Intact genes can be mutagenised, making it unnecessary to reclone the mutations before examining mutant phenotypes. The method has been applied to three residues around the N-terminus of the first a helix of the Klebsiella pneumoniue nitrogenase flavodoxin, which are predicted to form part of the phosphate-binding subsite. Surprisingly, most substitutions at Gly12, a highly conserved residue in the chain reversal preceding the a helix, appeared to be fairly stable in vivo and were found to retain some function. Substitutions at Lysl3, a surface residue which contributes to a patch of positive charge characteristic of the nitrogenase flavodoxins, had no major effect on stability or function. However, most substitutions at Thrl4, which is prcdicted to hydrogen bond to the phosphate of the prosthetic group FMN, were much more destabilising and grossly reduced function. The exceptions were Ala, Cys, Ser and Val, which suggests that the bulk of the residue at this position is critical.The power of oligonucleotide-directed mutagenesis in the analysis of protein structure and function has been greatly enhanced by the introduction of techniques which employ oligonucleotides containing randomised segments. Such degenerate oligonucleotides allow a range of substitutions to be isolated in one experiment, which in genetic analysis provides some safeguard against overinterpreting the effects of single mutations, and yields a range of mutant proteins from which the most interesting can be selected for biochemical and biophysical analysis. We have developed a powerful new method using degenerate oligonucleotides which we call 'roulette mutagenesis', and applicd it to the flavodoxin encoded by nifF in Klebsiella pneurnoniue.This flavodoxin is the immediate electron donor to component 2 of nitrogenase. It shows strong sequence similarity to other flavodoxins, which have a common structure comprising a parallel / 3 sheet sandwiched between a hclices (for review, see [ 2 ] ) . The crystal structure of the Synechococcus PCC 6301 (Aspergillus nidulans) flavodoxin has been used to roughly model the Klebsielln molecule [l]. 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 modifie...