Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe₂S₂] ferredoxins, products of PETF, FDX2–FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP⁺ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E_m = −321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (∼11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (−350 to −450 mV) that function as electron donors at reductive steps in various metabolic pathways (1–3). In photosynthetic organisms, the well studied ferredoxin (Fd⁴; the product of the PETF gene) is the most abundant iron-containing protein in the chloroplast and is central to the distribution of photosynthetically derived reductive power (4).The most well known Fd-dependent reaction is the transfer of electrons from photosystem I (PSI) to NADPH, catalyzed by Fd:NADP⁺ oxidoreductase (FNR). The NADPH produced by this reaction donates electrons to the only reductant-requiring step in the Calvin cycle and other steps in anabolic pathways that require NADPH as reductant. In addition, reduced Fd directly donates electrons to other metabolic pathways by interacting with various enzymes in the chloroplast. This includes Fd:thioredoxin reductase (FTR), which converts a light-driven electron signal into a thiol signal that is transmitted to thioredoxins (TRXs) present in the plastid as different types (or different isoforms). Once reduced, TRXs interact with specific disulfide bonds on target enzymes, modulating their activities (5). Other Fd targets include hydrogenase, which is responsible for hydrogen production in anaerobic conditions in green algae; glutamine-oxoglutarate amidotransferase in amino acid synthesis; nitrite and sulfite reductases in nitrate and sulfate assimilation, respectively; stearoyl-ACP Δ9-desaturase in fatty acid desaturation; and phycocyanobilin:Fd oxidoreductase in synthesis of phytochromobilin (6). Fd also functions in non-photosynthetic cells. Here, FNR catalyzes the reduction of Fd by NADPH produced in the oxidative pentose phosphate pathway, enabling Fd-dependent metabolism to occur in the dark (7, 8).The single-celled green alga, Chlamydomonas reinhardtii is an excellent reference organism for studying both metabolic adaptation to nutrient stress and photosynthesis (9–13). The Chlamydomonas genome encodes six highly related plant type ferredoxin genes (9). Until recently, only the major photosynthetic ferredoxin, Fd (encoded by PETF), which mediates electron transfer between PSI and FNR, had been characterized in detail (14).Many land plants are known to have multiple ferredoxins. Typically, they are differently localized on the basis of their function. Photosynthetic ferredoxins reduce NADP⁺ at a faster rate and are localized to the leaves, whereas non-photosynthetic ferredoxins are more efficiently reduced by NADPH and are localized to the roots. Arabidopsis thaliana has a total of six ferredoxin isoforms (15). Of these, two are photosynthetic and localized in the leaves. The most abundant, AtFd2, is involved in linear electron flow, and the less abundant (5% of the ferredoxin pool), AtFd1, has been implicated in cyclic electron flow (16). There is one non-photosynthetic ferredoxin located in the roots, AtFd3, which is nitrate-inducible. This protein has higher electron transfer activity with sulfite reductase in in vitro assays compared with other Arabidopsis ferredoxin isoforms, suggesting in vivo function of AtFd3 in nitrate and sulfate assimilation (15, 17). In addition, there is one evolutionarily distant ferredoxin, AtFd4, of unknown function with a more positive redox potential present in the leaves and two other proteins which are “ferredoxin-like” and uncharacterized (15). Zea mays has four ferredoxin isoforms, two photosynthetic and two non-photosynthetic (18). One of the non-photosynthetic isoforms is specifically induced by nitrite, suggestive of a role in nitrate metabolism (19). A cyanobacterium, Anabaena 7120, has two ferredoxins, vegetative and heterocyst type (by analogy to leaf and root types, respectively). The heterocyst type is present only in cells that have differentiated into nitrogen-fixing cells, indicating that this form may serve to transfer electrons to nitrogenase (20).We hypothesize that the presence of as many as six ferredoxin isoforms in a single-celled organism like C. reinhardtii allows for the differential regulation of each isoform and therefore the prioritization of reducing power toward certain metabolic pathways under changing environmental conditions. To test this hypothesis, expression of the genes (PETF and FDX2–FDX6) encoding the six ferredoxin isoforms in Chlamydomonas reinhardtii was monitored under various conditions in which well characterized ferredoxin-dependent enzymes are known to be expressed. In addition, we also analyzed the interaction of Fd and Fdx2 with several ferredoxin-interacting proteins, such as NiR, FNR, and FTR, and determined the kinetic parameters of the corresponding reactions.We found that each of the FDX genes is indeed differently regulated in response to changes in nutrient supply. In the case of FDX2 whose product is most similar to classical Fd, we suggest that it has specificity for nitrite reductase based on its pattern of expression and activity with nitrite reductase.     

MqsR, a Crucial Regulator for Quorum Sensing and Biofilm Formation, Is a GCU-specific mRNA Interferase in Escherichia coli

The mqsR gene has been shown to be positively regulated by the quorum-sensing autoinducer AI-2, which in turn activates a two-component system, the qseB-qseC operon. This operon plays an important role in biofilm formation in Escherichia coli. However, its cellular function has remained unknown. Here, we found that 1 base downstream of mqsR there is a gene, ygiT, that is co-transcribed with mqsR. Induction of mqsR caused cell growth arrest, whereas ygiT co-induction recovered cell growth. We demonstrate that MqsR (98 amino acid residues), which has no homology to the well characterized mRNA interferase MazF, is a potent inhibitor of protein synthesis that functions by degrading cellular mRNAs. In vivo and in vitro primer extension experiments showed that MqsR is an mRNA interferase specifically cleaving mRNAs at GCU. The mRNA interferase activity of purified MqsR was inhibited by purified YgiT (131 residues). MqsR forms a stable 2:1 complex with YgiT, and the complex likely functions as a repressor for the mqsR-ygiT operon by specifically binding to two different palindromic sequences present in the 5′-untranslated region of this operon.It has been reported that quorum sensing is involved in biofilm formation (1–4). mqsR expression was found to be induced by 8-fold in biofilms (5) and also by the quorum-sensing signal autoinducer AI-2, which is a species-nonspecific signaling molecule produced by both Gram-negative and Gram-positive bacteria, including Escherichia coli (6). It has been reported that induction of mqsR activates a two-component system, the qseB-qseC operon, which is known to play an important role in biofilm formation (6). Thus, it has been proposed that MqsR (98 amino acid residues) is a regulator of biofilm formation because it activates qseB, which controls the flhDC expression required for motility and biofilm formation in E. coli (6). However, the cellular function of MqsR has remained unknown.Interestingly, all free-living bacteria examined to date contain a number of suicide or toxin genes in their genomes (7, 8). Many of these toxins are co-transcribed with their cognate antitoxins in an operon (termed toxin-antitoxin (TA)² operon) and form a stable complex in the cell, so their toxicity is subdued under normal growth conditions (9–11). However, the stability of antitoxins is substantially lower than that of their cognate toxins, so any stress causing cellular damage or growth inhibition that induces proteases alters the balance between toxin and antitoxin, leading to toxin release in the cell.To date, 16 (12) TA systems have been reported on the E. coli genome, including relB-relE (13, 14), chpBI-chpBK (15), mazEF (16–18), yefM-yoeB (19, 20), dinJ-yafQ (21, 22), hipBA and hicAB (23, 24), prlF-yhaV (25), and ybaJ-hha (26). Interestingly, all of these TA operons appear to use similar modes of regulation: the formation of complexes between antitoxins and their cognate toxins to neutralize toxin activity and the ability of TA complexes to autoregulate their expression. The cellular targets of some toxins have been identified. CcdB directly interacts with gyrase A and blocks DNA replication (27, 28). RelE, which by itself has no endoribonuclease activity, appears to act as a ribosome-associating factor that promotes mRNA cleavage at the ribosome A-site (13, 29, 30). PemK (31), ChpBK (15), and MazF (32) are unique among toxins because they target cellular mRNAs for degradation by functioning as sequence-specific endoribonucleases to effectively inhibit protein synthesis and thereby cell growth.MazF, ChpBK, and PemK have been characterized as sequence-specific endoribonucleases that cleave mRNA at the ACA, ACY (Y is U, A, or G), and UAH (H is C, A, or U) sequences, respectively. They are completely different from other known endoribonucleases such as RNases E, A, and T1, as these toxins function as protein synthesis inhibitors by interfering with the function of cellular mRNAs. It is well known that small RNAs, such as mRNA-interfering cRNA (33), microRNA (34), and small interfering RNA (35), interfere with the function of specific RNAs. These small RNAs bind to specific mRNAs to inhibit their expression. Ribozymes also act on their target RNAs specifically and interfere with their function (36). Therefore, MazF, ChpBK, and PemK homologs form a novel endoribonuclease family that exhibits a new mRNA-interfering mechanism by cleaving mRNAs at specific sequences. Thus, they have been termed “mRNA interferases” (2).During our search for TA systems on the E. coli genome, we found that the mqsR gene is co-transcribed with a downstream gene, ygiT. These two genes appear to function as a TA system, as their size is small (98 residues for MqsR and 131 residues for YgiT) and their respective open reading frames are separated by 1 bp. In this study, we demonstrate that MqsR-YgiT is a new E. coli TA system consisting of a toxin, MqsR, and an antitoxin, YgiT. Moreover, we identify MqsR as a novel mRNA interferase that does not exhibit homology to MazF. This toxin cleaves RNA at GCU sequences in vivo and in vitro. The implication of this finding as to how this mRNA interferase is involved in cell physiology and biofilm formation will be discussed.     

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO₂⁻) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H₂O and D₂O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H₂O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D₂O than in H₂O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.Denitrification is an anaerobic respiration pathway found in bacteria, archaea, and fungi, in which ATP synthesis is coupled to the sequential reduction of nitrate (NO₃⁻) and nitrite (NO₂⁻) (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (1–3).³ The first committed step in this reaction cascade is the formation of gaseous NO by nitrite reductase (NiR), the key enzyme of this pathway. Two distinct classes of periplasmic NiR are found in denitrifying bacteria, one containing cd₁ hemes as prosthetic groups (4–6) and the other utilizing two copper centers to catalyze the one-electron reduction of nitrite (7). Copper-containing NiRs are divided into two main groups according to the color of their oxidized type 1 copper center (T1Cu), with shades ranging from blue to green (3, 7). NiR from Alcaligenes xylosoxidans subsp. xylosoxidans (NCIMB 11015, AxNiR), which is analyzed in this study, is a member of the blue CuNiR group. The blue and green subclasses show a high degree of sequence similarity (70%) (8) and have similar trimeric structures with each monomer (∼36.5 kDa in AxNiR) consisting of two greek key β-barrel cupredoxin-like motifs as well as one long and two short α-helical regions (7, 9).Each NiR monomer contains two copper-binding sites per catalytic unit. One is a T1Cu center, which receives electrons from a physiological redox partner protein and is buried 7 Å beneath the protein surface (10), and the other copper is a type 2 center (T2Cu), constituting the catalytically active substrate-binding site (11). The physiological electron donor for the blue NiRs are the small copper protein azurin (14 kDa) (7) and cytochrome c₅₅₁ (7, 12, 13). The T1Cu, which is responsible for the color of NiR, serves as the electron delivery center and is coordinated by two histidine residues as well as one cysteine and one methionine residue. The catalytic T2Cu, which like all T2Cu centers has very weak optical bands, is ligated to three His residues and an H₂O/OH⁻ ligand in the resting state. This H₂O/OH⁻ ligand is held in place by hydrogen bonds to the active site residues, Asp-92 (AxNiR numbering) and His-249, and gets displaced by the substrate during catalytic turnover (14). The T2Cu is located at the base of a 13–14-Å substrate access channel at the interface of two monomers with one of the three His residues being part of the adjacent subunit (15, 16). The two copper centers are connected by a 12.6-Å covalent bridge provided by the T1Cu-coordinating Cys and by one of the T2Cu His ligands (17, 18). This linkage has been suggested to constitute the electron transfer (ET) pathway from the T1Cu center to the catalytically active T2Cu center via 11 covalent bonds (19).Intramolecular ET from T1- to T2Cu has been extensively examined using pulse radiolysis studies (7, 19–24). In a variety of NiR species, ET could be measured, both in the presence and absence of substrate, with observed ET rate constants (k_ET(obs)) ranging from ∼150 to ∼2000 s⁻¹. According to the Marcus semi-classical ET theory (25), the redox potentials (E′₀, redox midpoint potential at pH 7.0) of the copper centers affect both the thermodynamic equilibrium and the ET kinetics. In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (E′₀ (T1Cu) ∼240 mV and E′₀ (T2Cu) ∼230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (≥150 s⁻¹) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H₂O/OH⁻ ligand and unable to bind nitrite (26, 27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7, 16, 25, 27–30). However, k_ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitrite-bound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16, 25, 30, 31). These rearrangements include subtle changes in the Cys-His bridge linking T1- and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref. 29). Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19, 27, 29, 32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14, 16, 19, 26, 27, 33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34). This finding, however, contradicts the pulse radiolysis results at room temperature (see above), and the apparent discrepancy between solution studies and x-ray crystallographic data collected at cryogenic temperature remains to be resolved.The one-electron reduction of nitrite to NO involves two protons according to the chemical net equation NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O, if the T2Cu is ligated by an H₂O molecule in the resting state rather than an OH⁻ ion. Although the exact enzymatic mechanism is still somewhat controversial (35, 36), one suggested reaction sequence is given in Scheme 1. The potential participation of active site residues in catalyzing the proton transfer (PT) steps has been investigated by studying the pH dependence of NiR under steady-state conditions as well as by pulse radiolysis. The trends obtained for k_cat and k_ET(obs), are similar with pH optima between 5.2 and 6, indicating the involvement of two amino acid residues (21, 22, 37). Asp-92 and His-249 have been proposed as acid-base catalysts (18, 21, 22, 28, 38), and the abrupt drop in rates at increasing pH may indicate that OH⁻ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35, 40, 41).⁴Open in a separate windowSCHEME 1.A potential reaction mechanism proposed for CuNiRs. Adapted from Ref. 36. Nitrite is shown to bind to the oxidized T2Cu as nitrous acid, thus involving the first protonation step. It coordinates to the oxidized T2Cu center in a bidentate fashion. Following inter-copper ET yielding a reduced T2Cu center, the initially deprotonated Asp-92 accepts a proton, which is subsequently transferred to the substrate. His-249 may be a potential source of this second proton. PT and ET reactions may be reversible and they may be concerted rather than sequential as suggested by the arrows. See text for further information.There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET. An extensive analysis of the solvent kinetic isotope effect (SKIE) has been employed as a means of determining whether solvent-exchangeable protons and/or water molecules play a rate-limiting role in the catalytic turnover and/or in inter-copper ET.     

Physiological and Genetic Analyses Leading to Identification of a Biochemical Role for the moeA (Molybdate Metabolism) Gene Product in Escherichia coli

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA⁻ phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.Molybdoenzymes play essential metabolic roles in most organisms from bacteria to plants and animals (34). All molybdoenzymes other than dinitrogenase contain molybdenum cofactor, which consists of a unique molybdopterin (MPT) complexed with molybdenum (1, 12, 23, 31, 34). In Escherichia coli, the biologically active form of the cofactor in molybdoenzymes is MPT guanine dinucleotide (MGD) (5, 22, 23). Synthesis of this cofactor in an active form requires transport of molybdate into the cell, activation of molybdate, synthesis of the MPT moiety, and incorporation of molybdate into MPT. Although molybdate transport and the various steps in the organic part of MGD biosynthesis are well characterized (17, 24, 33; see references 10, 22, and 23 for reviews), very little is known about the activation and incorporation of molybdenum into the cofactor (22).Mutants which are defective in molybdate metabolism can be isolated as chlorate-resistant mutants (8, 9). A large fraction of these mutants are pleiotropic for all molybdoenzyme activities in the cell, and these comprise the three genetic loci involved in MGD synthesis, moa, mob, and moeB (see references 10, 22, 29, and 31 for reviews). The mod gene products comprise the molybdate transport system through which molybdate is transported into the cell and the Mod⁻ phenotype can be suppressed by increasing molybdate concentration in the medium. The mog mutants which produced formate hydrogenlyase (FHL) activity containing the molybdoenzyme formate dehydrogenase-H (FDH-H) but not nitrate reductase activity was proposed to be defective in molybdochelatase (13, 32). This molybdochelatase is apparently required for production of active nitrate reductase and not for FDH-H.The moe operon codes for two proteins, and only the physiological role of the second gene product, MoeB protein, is known. The MoeB protein activates MPT synthase, which catalyzes the conversion of MPT precursor (precursor Z) to MPT by introducing the needed sulfur to which Mo is coordinated in the molybdenum cofactor (20, 22). The MoeB protein, MPT synthase sulfurylase, is the known S donor in the activation of MPT synthase. The physiological role of MoeA protein coded by the first gene in the two member moe operon is not known. Mutants which are defective in moeA (chlE [29]) produced about 6% of the wild-type levels of MPT (12), although no molybdoenzyme activity was found in these moeA mutants. Since the MoeB protein acts as an S donor in MPT synthesis, it is possible that the first gene product, MoeA protein, also has a similar role in linking S metabolism and Mo metabolism in the cell.During our analysis of molybdate transport-defective mutants, we identified a subgroup of chlorate-resistant mutants with a unique phenotype. Mutations in this class of mutants were mapped in the moeA gene at 18.6 min on the E. coli chromosome (3, 18). The MoeA⁻ phenotype was suppressed when the growth medium was supplemented with sulfide. In this report, we present the physiological and genetic characteristics of E. coli moeA mutants and propose a role for the MoeA protein in the activation of molybdenum by sulfurylation.(This work was presented at the International Symposium on Nitrogen Assimilation: Molecular and Genetic Aspects, 3 to 9 May 1997, Tampa, Fla.)