Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants

Abstract

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.

The nifY product of Klebsiella pneumoniae is associated with apodinitrogenase and dissociates upon activation with the iron-molybdenum cofactor

“…2A shows the position of accumulated component I antigens in extract from strain UN1655 (nifB). There is one predominant protein species in this strain that coelectrophoreses with purified Apo I (lane 3), and this strain has FeMo-co-activatable material (Table 1); therefore, this predominant band likely represents Apo I from this strain, which is in agreement with previous reports (3,4). The corresponding regions in Fig.…”

“…While it is certainly possible that other proteins may be involved in NifY dissociation in vivo, these results indicate that the in vitro dissociation of NifY from Apo I of K pneumoniae requires only the addition of FeMo-co. On the basis of the observation that the lowest band is the predominant species of component I antigen in the wild type and does not have NifY associated with it ( Fig. 2), it is tempting to speculate that this band is active component I and the middle band is an incompletely processed form, as has been suggested previously (3,4). As postulated previously, this species might have a FeMo-co at one site and one associated NifY molecule (3,19).…”

“…2), it is tempting to speculate that this band is active component I and the middle band is an incompletely processed form, as has been suggested previously (3,4). As postulated previously, this species might have a FeMo-co at one site and one associated NifY molecule (3,19). Such a hypothesis could predict that this band possesses component I activity, since the two FeMo-co sites may be functionally independent.…”

Involvement of GroEL in nif gene regulation and nitrogenase assembly

“…K. pneumoniae was normally grown in NFDM medium containing 0.2% ammonium acetate at 30°C (18). The two E. coli strains were grown similarly, but the medium was supplemented with 25 ,ug/ml each of L-proline, L-arginine, L-histidine, adenine, and thiamine.…”

“…The (5); pBZ147, containing the nipI promoter followed by nifHI'-'lacZ cloned in pMC1401 (5); pKT200 (6), containing an 8.1-kb EcoRI fragment including the groE operon from wild-type E. coli (13) cloned in pBR322; and pGR112 (34), containing an approximately 8-kb fragment of the K. pneumoniae nif cluster, including the nifLA operon. Transformation procedures were done as described before (18).…”