Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

Abstract

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P II homologs in the cell. P II is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P II binds ATP, we tested the hypothesis that removal of light would affect P II by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P II -mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P II under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.The P II family of regulatory proteins is one of the most broadly distributed in nature, being found in all three domains of life (1,20,39,51). P II was originally found to be involved in the regulation of glutamine synthetase (GS) activity by interaction with adenylyltransferase (ATase or GlnE, encoded by glnE) (63, 65). However, P II has subsequently been shown to regulate many other processes involving nitrogen assimilation and metabolism, by directly interacting with different proteins, termed P II receptors (51). In many organisms, more than one P II homolog has been identified. The "housekeeping" homolog is usually referred to as GlnB, encoded by glnB, with other secondary homologs named GlnK, GlnK 2 , GlnJ, GlnY, GlnZ, or Pz (1,12,47,69,78). In a few species of Archaea, a distinct group of P II proteins was named NifI, which is encoded by the nifH-linked gene (39).Because of its physiological diversity, the photosynthetic bacterium Rhodospirillum rubrum is a good model organism in which to study the role of P II . It has three P II homologsGlnB, GlnK, and GlnJ-that play distinct and overlapping functions by interacting with at least six receptors in the cell (71,78,81). Some of these are common to other bacteria, such as GlnE (ATase) that is involved in the regulation for GS activity, the two-component regulatory protein NtrB, and the ammonium transporter (gas channel) A...

Control of AmtB-GlnK Complex Formation by Intracellular Levels of ATP, ADP, and 2-Oxoglutarate

“…However, the highly conserved property of P II proteins to bind ATP or ADP and the observation that, in the AmtB-GlnK complex purified directly from ammonium-shocked cells, all the nucleotide-binding sites of GlnK are occupied by ADP, clearly implicate ATP/ADP binding as also playing a key role. Previous studies of ATP/ ADP pools following ammonium shock of nitrogen-limited cells have produced varied results, ranging from a very rapid drop in the intracellular ATP pool (33) to no detectable fluctuation in nucleotide pools (34,39). However, the culture conditions and the sampling methods used in all of those experiments were quite different from those used in this study.…”

“…Changes in GlnK Effector Concentrations-The inherent difficulties in measuring intracellular effector concentrations, in particular with regard to nucleotide pools, are well documented (37)(38)(39). It is also not possible to distinguish free and macromolecule-bound metabolites.…”

“…7B) (23). Gln 39 and Lys 58 are both located near the base of the T-loop, and hence, a consequence of this rearrangement will be a change in the conformation of the T-loop. The presence of ADP presumably reduces the conformational space of the T-loop, thereby favoring the novel extended conformation that promotes interaction with AmtB (23).…”

“…When GlnK is dissociated from AmtB, it is bound by ATP and 2-OG, both of which participate in coordination of Mg 2ϩ , for which they provide five oxygen ligands; the sixth ligand is provided by the conserved P II residue Gln 39 ( Fig. 7A) (24).…”

P II signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate

“…It was long considered that ATP could not play a regulatory role because its intracellular concentration is typically 1-5 mM, whereas the affinity of P II proteins for the nucleotide is in the micromole range (K d ∼50 μM) (11,12). However, the subsequent recognition that ADP is also a physiological effector (10) led to a reevaluation of the role of nucleotides, and a number of studies concluded that P II proteins might also act as sensors of cellular energy status, as reflected by fluctuations in the ATP/ADP ratio (13)(14)(15)(16)(17)(18).…”