The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighbor of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potential channel for entry/exit of substrates/products and for accepting FeMo cofactor during MoFe-protein maturation. Several of the eight mutant strains constructed were capable of good diazotrophic growth and also contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively charged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofactor-deficient, apo-MoFe protein. The wide range of nitrogen-fixation phenotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substituted strain, DJ788 (alpha-277His). This strain, although unable to fix N2 and grow diazotrophically, elaborates an altered alpha-277His MoFe protein that catalyzes the reduction of the alternative substrates, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the alpha-277His MoFe protein is incapable of reaching the more-reduced redox levels required for nitrogen fixation. Under nonsaturating CO concentrations, the alpha-277His MoFe-protein-catalyzed reduction of C2H2 showed sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity among two C2H4-evolving sites and indicates the presence of three sites, which can be simultaneously occupied, on the MoFe protein. Similar kinetics were not observed for alpha-277His MoFe-protein-catalyzed reduction of either HCN or HN3 with nonsaturating CO levels, indicating that these substrates are unlikely to share common binding sites with C2H2. Further, CN- did not induce cooperativity in C2H2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by changes in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indicate the importance of the alpha-277 residue in catalysis and the delicate control exerted on the properties of bound FeMo cofactor by its polypeptide environment.

Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 M. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the conversion of isocitrate to ␣-ketoglutarate, with concomitant production of NADH and carbon dioxide (3, 15). In Escherichia coli, ICDH activity is regulated by posttranslational modification involving a phosphorylation of serine 113 within the homodimeric protein (16,17,34,36). This reaction is catalyzed by the AceK protein, isocitrate dehydrogenase kinase/phosphatase, to inactivate ICDH under conditions when the cell is grown on acetate or fatty acids. AceK also serves as a protein phosphatase that restores ICDH activity under alternative cell growth conditions when glucose or its saccharide precursors are present. The modulation of ICDH enzyme activity in the cell aids in maintaining the optimal amounts of TCA cycle intermediates, since this enzyme is at the branch point for carbon flow to the glyoxylate bypass pathway (3,14). In the competing reactions, isocitrate lyase converts part of the isocitrate pool to glyoxylate and succinate, while malate synthase then combines glyoxylate with acetyl coenzyme A to form malate. Thus, when E. coli is grown on acetate or its direct precursors, the glyoxylate bypass reactions makes it possible for the cell to generate four-carbon compounds needed for biosynthetic reactions while also balancing its needs for energy via TCA cycle-derived NADH and FADH.Little is known about the control of isocitrate dehydrogenase (icd) gene expression in E. coli under different cell growth conditions. Early studies by Gray et al. demonstrated that ICDH enzyme activity varied over a 10-fold range depending on the availability of oxygen and the composition of the cell growth mediu...

SummarySuccinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC-lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at ¹205 bp and ¹119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the ¹35 and ¹10 regions of the sdhC promoter. A fourth ArcA site is centred at þ 257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.

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