Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.

Abstract

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron-and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphatecontaining homodimer that catalyzes the formation ofL-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metaflocluster formation. The recent discovery of nifSlike genes in non-nitrogen-fiing organisms also raises the possibility that the reaction catalyzed by NIIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metailocluster formation.The reduction in both A. vinelandii nitrogenase component protein activities as a result of nijS deletion, which has also been reported for Klebsiella pneumoniae (6), could not be attributed to a regulatory effect (3). Thus, because both Fe protein and MoFe protein activities were affected by deletion of nijf , and the common feature of both component proteins is that they contain metalloclusters, we have considered that NIFS might be involved in the acquisition or mobilization of the inorganic Fe or sulfur required for metallocluster formation. This possibility is supported by the observation that cell pellets of diazotrophically grown A. vinelandii nifS mutants are pale tan rather than the characteristic dark brown of wild type. The dark color of diazotrophically grown wild-type A. vinelandii is attributed to the metalloclusters contained within the nitrogenase component proteins that form a substantial portion of the soluble protein fraction.As a strategy to elucidate the function of the NIFS polypeptide, we chose to produce large amounts of it in Escherichia coli to facilitate its purification and biochemical characterization. In the present study, we have purified the NIFS protein, demonstrated that a specific reaction is catalyzed by NIFS, and proposed a biochemical function for NIFS activity in relation to metallocluster formation. In addition, we discuss the possibility that the reaction catalyzed by NIFS represents a universal mechanism for the mobilization of the sulfur required for metallocluster formation. Many proteins that have important electron tr...

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

“…Incubating NifL (1.4 nmol/50 l) with the NifS enzyme from A. vinelandii (0.37 nmol/50 l) in the presence of ferrous iron, cysteine, and ATP did not relieve inhibition (see Materials and Methods; data not shown). Under these conditions, NifS is known to catalyze the formation of Fe-S clusters in both components of nitrogenase and in other proteins (26,(52)(53)(54). We could not synthesize NifL under conditions appropriate for NifS activity because the concentrations of ferrous iron required were inhibitory to the function of the coupled system (46).…”

“…NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9).…”

“…NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35).…”

“…NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35). NifL, which is a negative regulator, inhibits NifA activity in vivo in response to molecular oxygen and/or combined nitrogen (22,32).…”

HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a [4Fe–4S]‐containing oxygen‐responsive transcription regulator that anaerobically activates FNR‐dependent Class I promoters via an enhanced AR1 contact

“…Bovine serum albumin served as the standard, with a correction factor of 1.1 being applied to allow for the difference between this standard and a reference sample of GST-HlyX (analysed by Alta Bioscience, University of Birmingham). NifS protein was purified from E. coli BL21( DE3) containing the nifS expression plasmid pDB551 (Zheng et al, 1993).…”