Progress has been made in the understanding of photorespiration and related proteins (Rubisco, glycolate oxidase and glycine decarboxylase) in the context of recent structural information. Numerous shuttles exist to support transamination, ammonia refixation and the supply or export of reductants generated or consumed (via malate-oxaloacetate shuttles) in the photorespiratory pathway. A porin-like channel that is anion selective represents the major permeability pathway of the peroxisomal membrane.
Addresses
DBMS, Laboratoire de Physiologie Cellulaire Végétale, CEA Grenoble et Université Joseph Fourier, 17 rue des martyrs, F 38054 Grenoble, Cedex 9, France
*e-mail: [email protected]
Current Opinion in Plant Biology1999, 2:214–222 http://biomednet.com/elecref/1369526600200214 © Elsevier Science Ltd ISSN 1369-5266
Abbreviation
RuBP ribulose-1,5-bisphosphate
Introduction
The prime function of the C2 oxidative photosynthetic car-bon cycle — inappropriately named ‘photorespiration’ [1•] — is to salvage glycolate-2-P produced continuously in the light by the oxygenase activity of ribulose-1,5-bisphos-phate carboxylase/oxygenase (Rubisco). In leaves under ambient conditions the rate of oxygenation to carboxylation has been estimated as high as 0.4. Low intercellular con-centrations of CO2, as may occur, for example, under water stress (e.g. whenever the stomata are closed), can result in even higher ratios. Given the voluminous literature on pho-torespiration [2,3], in this short review we merely highlight recent advances in this topic, laying emphasis on a few pho-torespiratory enzymes (Rubisco, glycolate oxidase and glycine decarboxylase) and molecular traffic between per-oxisomes, chloroplasts, and mitochondria.
The photorespiratory pathway
The value of numerous mutant plants (Hordeum, pea, and Arabidopsis thaliana) in the exquisite elucidation of the mech-anism of photorespiration and its relationships with CO2 fixation and amino acid metabolism has been highlighted by several groups (see [3] for a full list). These mutants were unable to survive in air, but could thrive in atmospheres con-taining a high concentration of CO2(or low [O2]).
The recycling of glycolate-2-P into glycerate-3-P via the photorespiratory pathway and then further to ribulose-1,5-bisphosphate (RuBP) is not only a very costly reaction, it also requires a large machinery consisting of 16 enzymes and more than six translocators, distributed over the chloro-plast, peroxisome and mitochondrion in close proximity to
each other. In the course of this pathway two molecules of glycolate-2-P are metabolized to form one molecule each of glycerate-3-P and CO2 and these carbon compounds are used immediately for the regeneration of RuBP via the Benson–Calvin cycle (C3 cycle) without the net synthesis of triose phosphate. Once glycolate-2-P is formed, the pho-torespiratory cycle works forward to convert all the carbon diverted out of the C3 cycle back to photosynthesis as rapidly as possible [3]. Indeed, several reactions occuring in chloroplasts and peroxisomes strongly favor product forma-tion. Obviously, although very little is known about the feed-back mechanisms that might operate in photorespira-tion [4], the most important control step is at the level of competition between O2and CO2for binding to Rubisco.
In C3plants the C2cycle is operating in the photosynthet-ically active mesophyll cells. In C4 plants the C2 cycle operates in the bundle sheath cells [5]. Using two geneti-cally modified C4plants, a mutant of Amaranthus edulisthat is deficient in PEP carboxylase and a transgenic plant Flaveria bidentis which has reduced levels of Rubisco, Marocco et al [6•] have demonstrated that when the C
4 plant is ineffective in concentrating CO2 in the bundle sheath cells there is a marked increase in photorespiration and when the C4plant exhibits low levels of Rubisco there is a marked increase in bundle-sheath CO2 leakage. This observation provides definitive evidence that photorespi-ration is insignifiant in C4plants because they are capable of concentrating CO2in the bundle-sheath cells leading to the suppression of the oxygenase reaction of Rubisco.
Functioning of key enzymes involved in
photorespiration
Few enzymes involved in this cycle have been studied carefully. Only Rubisco, glycolate oxidase and a sophisti-cated set of proteins involved in glycine cleavage (glycine decarboxylase system) have been studied in an exhaustive manner. For this reason we have chosen to focus on a restricted set of enzyme systems.
Mechanism of Rubisco: the triggering of photorespiration
Rubisco is present at a tremendous concentration in the stroma of the chloroplasts (~0.2 g ml–1) stromal extract and catalyses both the carboxylation (the enzyme exhibits a low catalytic rate constant, 3.5 sec–1) and the oxygenation of ribulose-1,5-bisphosphate [7–9,10••]. The two reactions involve the competition of molecular CO2 with O2 for the 2,3-enediol(ate) form of RuBP which is first generated at the active site of the enzyme. At any given [CO2][O2], the frac-tional partitioning of RuBP between the carboxylation and oxygenation pathways is governed by the relative reactivity of the enzyme-bound 2,3-enediol(ate) toward CO2 and O2[10••]. From biochemical analyses of Rubisco purified from several species, including photosynthetic bacteria,
Biochemical dissection of photorespiration
differences in specificity towards the substrates CO2and O2: evolutionary pressures seem to have directed Rubisco towards more efficient utilization of CO2 [10••,11]. Rubisco from cyanobacteria, green algae and higher plants is assem-bled from eight large (L) subunits and eight small (S) subunits (four dimers of L subunits surrounded by two tetramers of S subunits; (L2)4(S4)2) [12], whereas Rubisco from Rhodospirillum rubrum (a nonsulfur purple bacteria) consists of only two large subunits (L2). The large subunit from spinach can be divided into two domains, an amino terminal domain and a carboxy-terminal α/β-barrel domain. Two active sites are located at the interface of the L-sub-units in the L2 dimer (‘head to tail’ arrangement). The catalytic center is mostly situated at the carboxy-terminal end of the α/β-barrel. The enhancement of catalytic rate by S subunits can only mediated through induced conforma-tional changes in catalytic subunits because S subunits are far removed from the active site [13].
A large number of crystal structures of Rubiscos from var-ious sources including Rhodospirillum rubrum, Synechococcus, and spinach have been reported along with a variety of ligands (see [10••,], for a full list) and this, in synergy with biochemical investigations, led to a careful dissection of the carboxylation pathway. The carboxyla-tion of RuBP involves multiple discrete steps and associated transition states: removal of ligands such as 2-carboxyarabinitol 1-phosphate from the inactive enzyme form (this process occurs slowly by simple dissociation, or rapidly when catalysed by the enzyme Rubisco activase); carbamylation of the ε-amino group of Lys201 (spinach) residue in the active site by an activator CO2 molecule; stabilization of this protein-bound carbamate by mon-odentate coordination to Mg2+ (three water molecules, Asp203 and Glu204 complete the octahedral coordination sphere around this metal ion); binding of RuBP: it is ori-ented in the active site with the Siface of the C-2 (and C-3) accesible to the bulk solution (for an explanation, see [10••]); removal of the C-3 proton of RuBP to effect enolisation (the deprotonating agent is still not identi-fied); addition of CO2 to the Si face of C-2 and water to the Siface of C-3 to yield the six-carbon hydrated inter-mediate (2′-carboxy-3-keto-D-arabinitol 1,5 bisphosphate); and carbon–carbon cleavage between C-2 and C-3 to form two glycerate-3-P molecules] [10••,14••]. Higher resolution structures of both the Synecococchus[15] and of the spinach enzyme [12,14••] demonstrated the key role of the carbamate on K201 in the carboxylation pathway. The role of Rubisco activase in limiting steady state photosynthesis has been examined using transgenic plants with reduced levels of activase [16,17]. It was con-cluded that Arabidopsis grown under high and low irradiance does not contain Rubisco activase in great excess of the amount required for optimal growth [16]. In addition, a phase in the activation of Rubisco that repre-sents the activation of the 2-carboxy arabinitol 1 phosphate inhibited form of Rubisco was discerned [17].
glycerate-3-P (formed from C-3, C-4 and C-5 of RuBP), and glycolate-2-P. The oxygenation pathway has not been dissected as deeply as the carboxylation counterpart [10••]. Very likely the oxygenation pathway is similar to the car-boxylation pathway although the putative key labile intermediate (2-peroxy-3-ketoarabinitol 1,5-bisphosphate) [10••] postulated through the exquisite characterization of two-different site directed mutants (E60→Q, K334→A) [18,19], has never been characterized so far. This reaction may be an inevitable consequence of Rubisco’s inability to protect its ene-diolate reaction intermediate from O2. Indeed, this notion is supported by the failure of numerous efforts to eliminate selectively its oxygenase activity by genetic manipulation. The partitioning of RuBP between the carboxylation and oxygenation pathways is sensitive to the active site microenvironment and does not involve large movements within the structure [10••]. Given the structural similarity of the two alternative substrates CO2 and O2 and the large difference in their concentration within the chloroplasts, it is clear that Rubisco influences the selectivity for CO2in some way [12].
Glycolate oxidase
Glycolate oxidase (an octamer composed of identical sub-units of approximately 40 kDa) is one of the very few peroxisomal proteins for which a high resolution crystal structure is available [20•]. The enzyme from spinach crys-tallizes in an octameric form and the subunit contains an eight-fold β/α barrel motif corresponding to the flavin mononucleotide (FMN) domain which is also found in other FMN-dependent enzymes. The irreversible reaction catalysed by the enzyme can be divided into two half-reac-tions. First glycolate is oxidised by the flavin which is deeply burried in the barrel. In the second part FMN is reoxidized by O2 to produce H2O2 which is, in turn, decomposed by catalase (a heme-containing enzyme). The active site is formed by the loops at the carboxy-terminal end of the β-strands in the barrel. The amino acids involved in the structure of the active site have been stud-ied [21]. Thus, the replacement of Trp108 by Ser led to dramatic effects on both the Kmof substrate as well as on the turnover number indicating that this amino acid is of crucial importance in catalysis and in determining the sub-strate specificity of glycolate oxidase. Likewise Tyr24 is involved in binding of the substrate by way of hydrogen-bond formation between its hydroxyl group and the carboxylate group of the substrate molecule.
During the course of glycolate oxidation, proceeding in an irreversible way, huge amounts of hydrogen peroxide are released in the peroxisomes. Most of the hydrogen perox-ide is degraded by catalase, but the high Km (millimolar range) for the enzyme could result in low harmful residual concentrations diffusing into contact with the inner surface of the limiting peroxisomal membrane which contains an ascorbate peroxidase [23]. Transgenic tobacco with 0.05 to 0.15 times the catalase activity of wild-type has been reported [24], and it was shown that under high photores-piratory conditions necrotic lesions were produced in leaves owing to dramatic accumulation of H2O2.
Reaction catalysed by the glycine
decarboxylase multienzyme complex
Rapid glycine oxidation, which requires the functioning of two enzymatic complexes (glycine decarboxylase and ser-ine hydroxymethyltransferase) working in concert, is a key step of the C2 cycle because it results in the conver-sion of a two-carbon molecule into a three-carbon molecule that thereafter, could be reintroduced in the C3 cycle [25]. The glycine decarboxylase multienzyme com-plex, present at tremendous concentration in the matrix of
plant mitochondria, has been purified and, like its mam-malian counterpart, contains four different component enzymes designated as the H-protein (a monomeric lipoamide-containing protein, 14 kDa), P-protein (a homodimer containing pyridoxal phosphate, 200 kDa), T-protein (a monomer acting in concert with folate [5,6,7,8-tetrahydropteroylpolyglutamate; H4PteGlu], 45 kDa) and L-protein or lipoamide dehydrogenase (a homodimer containing flavin adenine dinucleotide [FAD] and a redox active cystine residue, 100 kDa) [25]. All the protein components of the glycine decarboxylase system dissociate very easily and behave as non-associated pro-teins following mitochondrial inner membrane rupture after several cycles of freezing and thawing.
The H-protein acts as a mobile co-substrate that com-mutes between the other three proteins (Figure 1). Its lipoyl moiety (attached by an amide linkage to the ε-amino group of a lysine residue [Lys63in the 131 amino acid pea H-protein; 26] which is located in the loop of an hairpin configuration [27]) undergoes a cycle of reductive methy-lamination, methylamine transfer and electron transfer. The reaction commences with the amino group of glycine
Figure 1
Outline of the reactions involved in oxidative decarboxylation and deamination of glycine in plant mitochondria. Glycine decarboxylase consists of four different component proteins: P, T, H, and L. H-protein is a 14.1 kDa monomer that plays a pivotal role in the reaction mechanism, as it interacts sequentially with each of the other three proteins through its lipoic acid cofactor bound to a lysine residue. The P-protein component (this enzyme has a Mrof 210,000 and is a homodimer of 105,000 Mrpolypeptides) catalyses the decarboxylation of glycine and the reductive transfer of the resultant methylamine moiety to the lipoyl-lysine (lipoamide arm) of the H-protein. The lipoate cofactor is located in the loop of a hairpin configuration, but following methylamine transfer, it is pivoted to bind into a cleft at the surface of the H-protein. The lipoamide-methylamine arm is, therefore, not free to move in the solvent. The lipoamide-methylamine arm is then shuttled to the T-protein (a 45,000 Mr
monomer) where the methylene carbon is transferred to tetrahydrofolate (H4FGlu5), producing CH2-H4FGlu5and releasing the amino nitrogen as NH3. Finally, the reduced lipoamide resulting from this transfer is reoxidized by the FAD coenzyme bound to the L-protein (a homodimer of 60,000 Mr polypeptides), with the sequential reduction of FAD and NAD+. SHMT, serine
hydroxymethyltransferase is involved in the recycling of CH2-H4FGlu5to H4FGlu5.
HS HS NH
O NADH
NAD
H4FGlu5 CH2 H4FGlu5
NH3
CO2
S S
N O
H
O NH CH2
S
SH NH3
NH3
COO H2C
H2C
COO
NH3
H2C
COO
NH3
HOH2C
P
L
T
SHMT
H
oxH
metH
redP-protein. The carboxyl group of glycine is lost as CO2and the remaining methylamine moiety is passed to the lipoamide cofactor of the H-protein; when it is oxidized the lipoyl moiety is free to move in the solvent and is allowed to visit the active site of the P-protein. The rapid methylamination of the H-protein is half-saturated at micromolar concentrations of H-protein (Km H-protein = 9 µM; Vmax = 5 µmol mg–1protein min–1). During the course of the reductive methylamination, the lipoamide-methylamine arm formed rotates to interact readily with several specific amino acid residues located within a cleft at the surface of the H-protein; the methylamine group linked to the distal sulfur of the dithiolane ring is tightly bound by ionic and hydrogen bonds to residues Glu14, Ser12, and Asp67, whereas the carbon atoms of the lipoamide arm interact through van der Waals contacts with hydrophobic residues [27,28].
Such a situation locks the methylamine group into a very stable conformation preventing the non-enzymatic release of NH3 and formaldehyde which would otherwise take place due to nucleophilic attack by OH– of the carbon atom bearing NH2 group until the reaction with H4PteGlun and T-protein takes place. In the absence of H4PteGlunin the incubation medium the T-protein causes a change in the overall conformation of the H-protein, leading to the release of the lipoamide-methylamine arm from the cleft at the surface of the H-protein. These cir-cumstances, therefore, favour, the nucleophilic attack by OH– of the carbon atom bearing NH
2 group; NH3 and formaldehyde accumulate slowly in the incubation medi-um and the lipoamide arm becomes fully reduced (Figure 2). On the other hand, in the presence of H4PteGlun formaldehyde does not accumulate because the methylamine group undergoes a preferential nucle-ophilic attack by the N-5 atom of the pterin ring of H4PteGlun: NH3and CH2H4PteGlun; accumulate rapidly in the medium concomitantly with the reduction of the lipoamide arm (Figure 2).
Plant mitochondria possess a powerful NAD-dependent for-mate dehydrogenase [29]. They also possess a formaldehyde dehydrogenase. These enzymes are not believed to be involved in the main route of carbon flow through the glyco-late pathway. They could serve as rescue reactions, neutralising the harmful effect of formaldehyde molecules produced by the glycine cleavage system in a non-controlled reaction. Finally, the L-protein (dihydrolipoamide dehydro-genase) catalyses the regeneration of the oxidised form of lipoamide with the sequential reduction of FAD and NAD+. This rapid oxidation is half-saturated at micromolar concen-trations of H-protein (Km reduced H-protein = 20 µM). In green leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihy-drolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase, L-protein of glycine decarboxylase) [30] and this raises some interesting questions about the regulation of
enzyme associated with different complexes. For example, the distribution of L-protein among complexes may rely upon various metabolic situations.
In leaf mitochondria, the major function of serine hydrox-ymethyltransferase (SHMT, a 220 kDa homotetramer) is to recycle CH2H4PteGlunproduced by the T-protein activ-ity to H4PteGlun, to allow the continuous operation of the glycine-oxidation reaction [31]. This reaction is perma-nently pushed out of equilibrium towards the production of serine and CH2H4PteGlun [32] that is the forward motion of the photorespiratory cycle. The T-protein and SHMT do not associate and the reaction intermediates are not directly transferred through a channeling mechanism from the active site of T-protein to that of SHMT.
Photorespiratory nitrogen cycle
Quantitatively, the conversion of glycine to serine in the C2cycle is probably the most important metabolic process that liberates ammonia within the mesophyll cells. Nitrogen is inserted into the C2cycle through a transami-nation step in the peroxisome catalysed by a glutamate:glyoxylate aminotransferase [2]. Ammonia liber-ated in the matrix of mitochondria during the course of glycine oxidation diffuses rapidly to the chloroplast where it is used, with a very high affinity, by glutamine syn-thetase catalysing the ATP-dependent conversion of glutamate to glutamine [33]. Indeed Mattson et al. [34] demonstrated that in barley mutants with reduced gluta-mine synthetase the rate of ammonia emission correlated with the concentration of ammonia in the leaves. In bacte-rial glutamine synthetase, the active site is located between adjacent subunits and structural models for the reaction mechanism based on five crystal structures of enzyme–substrate complexes have shown that the reaction occurs in two steps. First ATP binds to the active site fol-lowed by glutamate to yield γ-glutamyl phosphate and ADP. Then NH4+binds to the active site, which, after los-ing a proton, attacks the γ-glutamyl phosphate with the liberation of glutamine and phosphate [35,36]. Whether a similar mechanism also operates in eukaryotic octameric glutamine synthetase is still a matter of debate. It is clear now from the analysis of barley mutants deficient in gluta-mine synthetase that the chloroplastic isoform is directly involved in the reassimilation of ammonia released during the process of photorespiration [33]. On the other hand, the cytoplasmic isoform is localized in the vascular system and the phloem companion cells of the leaf [37,38], thus precluding any role in photorespiration.
glutamate:glyoxylate amino transferase in exchange for 2-oxoglutarate. Arabidopsis contains two expressed genes for this enzyme (Glu1 and Glu2) situated on different chromosomes. Glu1 plays a major role in photorespiration in Arabidopsis, as has been determined by the characteriza-tion of mutants deficient in this form [39]. Glu2 may play a major role in primary nitrogen assimilation in roots. The enzyme (monomeric with an Mr of ~160 kDa) contains one FMN and one {3Fe-4S} cluster per molecule [40]. The assay of this enzyme activity has been greatly facilitated by the use of methyl viologen as a source of reductant [41] which is recognized by the ferredoxin-binding site con-taining two critical lysine and arginine residues [41,42].
An interesting point recently raised by Migge et al. [43•] was that key enzymes of the photorespiratory nitrogen cycle were not affected either by growing plants in elevat-ed CO2 partial pressure (short-term exposure) or by the rate of photorespiratory ammonium production, thus allowing C2-cycles and nitrogen-cycles to take place imme-diately following exposure to normal air.
Molecular traffic
During the course of photorespiration, massive traffic of various molecules occurs between different cell organelles. Unfortunately, the major characteristics of the transport proteins (reconstitution of the transporter into liposomes, kinetic parameters, multisubunit nature, high-resolution structures, and multifaceted regulation) catalysing sub-strate travel through membranes to fulfil photorespiration have been poorly studied. We must say that it is always a real ‘tour de force’ to reconstitute a transporter into lipo-somes in an active form.
NH3and CO2movement
The NH4+(and/or NH3) released during glycine oxidation passes through the inner membrane of mitochondria and chloroplasts. Whether this passage occurs by simple diffu-sion, or is brought about by specific ion channels or translocators is still a matter of debate. In order to maintain ammonia emission close to zero when carbon assimilation is strongly limited by stomatal closure under drought con-ditions, we should expect a specific mechanism to divert
Figure 2
Current Opinion in Plant Biology
Proposed model for the reaction catalysed by the T-protein. (a)In the absence of H4FGlunin the incubation medium the T-protein causes a change in the overall conformation of the H-protein, leading to the release of the lipoamide-methylamine arm from the cleft at the surface of the H-protein (see Figure 1). Such a situation favours, therefore, the nucleophilic attack by OH–of the carbon atom bearing the NH
2group;
support of this suggestion a gene from Arabidopsis for a high affinity ammonia transporter has been identified [44]. We can speculate, therefore, the presence of a specific ammonia transporter on the inner membrane of the chloro-plast envelope.
Likewise, one of the major unresolved aspects of the inner membranes of mitochondria and chloroplasts in all eukary-otes concerns the CO2permeability of the membranes. In other words it is not known which carbon inorganic species (CO2, HCO3-) is transported in cell organelles. In this con-nection Rolland et al. [45], using a mutant of Chlamydomonas reinhardtii, have suggested the existence of a specific pro-tein within the plastid envelope which promotes inorganic carbon uptake into chloroplasts. Very likely, this protein is the product of the chloroplast ycb10 gene which has been localized in the inner membrane of the plastid envelope. The disruption of this gene in Chlamydomonasusing biolis-tic transformation was correlated with a decrease in CO2-dependent photosynthesis and a reduced affinity of the CO2and HCO3-uptake system for their substrates.
Chloroplast transporters
Glycolate must move from the stroma to the peroxisome across the inner envelope membrane and D-glycerate must go in the opposite direction. Experiments with intact chloroplasts have shown that a single carrier-type trans-porter is responsible for the movement of both glycolate and D-glycerate across the chloroplast inner envelope membrane. This transporter was solubilized by treatment of the chloroplast inner membrane by sodium cholate and reinserted into artificial vesicles [46] . The glycolate/glyc-erate transporter is interesting because it does not catalyse a strictly coupled substrate exchange (however, glycolate and D-glycerate stimulate one another’s transport from the opposite side of the membrane); unidirectional influx or efflux also occurs as a proton symport or hydroxyl antiport. This flexibility allows the amount of glycerate returning to the chloroplasts to be only half that of the glycolate released from the chloroplasts.
During the course of photorespiration, 2-oxoglutarate is massively imported into the chloroplasts, and glutamate, deriving from the glutamine synthetase/glutamate synthase cycle, is exported towards the peroxisome. Two different dicarboxylate antiport systems with overlapping substrate specificities are involved in this process. The 2-oxoglu-tarate/malate translocator imports 2-oxoglutarate in exchange for stromal malate, whereas export of glutamate from the chloroplast in exchange for malate is catalysed by the glutamate/malate translocator. Malate is, therefore, the counterion for both translocators, resulting in 2-oxoglu-tarate/glutamate exchange without net malate import [47]. A cDNA clone encoding the spinach chloroplast 2-oxoglu-tarate/malate translocator has been obtained by Weber et al. [48]. The predicted protein with an apparent molecular mass of 45 kDa contains a 12-helix motif and probably
porters of organellar origin, including mitochondria, that have 5–7 transmembrane helices functioning as dimers. The transit peptide of this translocator is extremely long although its import characteristics closely resemble those of other inner envelope membrane proteins. The 2-oxoglu-tarate/malate translocator could be functionally expressed in the fission yeast Schizosaccharomyces pombe and subse-quent reconstitution of the recombinant protein in liposomes demonstrated definitively that this translocator mediates the exchange of 2-oxoglutarate with malate. Obviously the glutamate/malate carrier, which also plays a critical role in the recycling of ammonia during the course of photorespiration, requires an exhaustive study in order to understand precisely the interplay of both carriers working in concert.
Mitochondria transporters
The rate of glycine oxidation demands that green leaf mito-chondria support a phenomenal rate of glycine transport (0.8–1.6 µmol min–1mg–1 protein). In the course of glycine decarboxylation and deamination, one molecule of serine leaves the mitochondrion and two molecules of glycine are taken up. For the present, we have to admit that the details of glycine and serine transport in green leaf mitochondria remain a mystery and the question as to whether both glycine and serine are transported by a single protein or by two different ones cannot be answered at present.
The conversion of hydroxypyruvate to glycerate in the per-oxisomal matrix requires NADH as reductant. Peroxisomes are, therefore, dependent on the supply of reducing equiv-alents from the cytoplasmic compartment. On the other hand, NADH produced during the course of glycine oxida-tion is reoxidized very rapidly by oxaloacetate owing to the tremendous excess of NAD+-linked malate dehydrogenase in the matrix space. The malate produced from this reac-tion is removed from the mitochondria in exchange for cytosolic oxaloacetate by a specific oxaloacetate transporter. Peroxisomes are supplied, therefore, with reducing equiva-lents not by direct uptake of NADH but by indirect transfer via this malate–oxaloacetate shuttle [49]. A very powerful phthalonate-sensitive oxaloacetate carrier has been characterised in all the plant mitochondria isolated so far [50]. This rapid phthalonate-sensitive uptake of oxaloac-etate, which plays an important role in the C2 cycle, is half-saturated at micromolar concentrations of oxaloacetate (KmOAA= 5 µM; Vmax= 2 µmol min–1per mg of protein). The activity of this carrier appears to be high enough to account for in vivocarbon fluxes through the inner mito-chondrial membrane. The purification and functional reconstitution, as well as the completion of detailed kinetic analyses, of this specific transporter should be undertaken.
Porin of peroxisomal membrane
been proposed that this membrane contains a slightly anion-selective channel-forming component, in accordance with its physiological function and distinct from other known eukaryotic porins [51,52,53•]. For example, its sin-gle-channel conductance of about 300 pS (in 1 M KCl) is one order of magnitude lower than that of the mitochondr-ial porin. The narrow diameter (0.6 nm) of this pore-forming protein restricts the diffusion to anions (gly-colate, glycerate, etc.). The characterization of a binding site for dicarboxylate anions inside the peroxisomal chan-nel, however, is puzzling. It is possible, in analogy with inducible porins which have been characterized in some gram-negative bacteria, that this binding site confers rather selective properties to this peroxisomal channel, preventing the diffusion of highly reactive intermediates of peroxiso-mal metabolism, such as glyoxylate and H2O2[53•].
Conclusions
It has been claimed that Rubisco behaves as a ‘Schizophrenic’ enzyme because of its inability to protect it’s ene-diolate reaction intermediate from O2 [13]. This unfair statement should be reconsidered [1•], however, because several groups have demonstrated that photorespi-ratory metabolism can prevent the formation of the excited triplet state of chlorophyll and excess reactive O2 species (superoxide radicals and singlet oxygen) which necessarily occur in sunlight when CO2, the final electron acceptor, is lacking [54]. In other words photorespiration, a very ‘waste-ful’ process, in concert with other reactions including a cycle utilizing monodehydroascorbate reductase, dehy-droascorbate reductase and glutathione reductase (Halliwell-Asada cycle) alleviates the damage that oxygen radicals can cause in green leaves [55,56]. Wasteful and use-ful are not necessarily incompatibles and very likely Rubisco is more ‘clever’ than we thought because when stomata are closed (the CO2concentration of the intercel-lular space of the leaves drops to the CO2 compensation point) C3- and C2-cycles operate in perfect synchrony to prevent excessive reduction, and, therefore, photoinactiva-tion, of the chloroplast electron transport chain [55].
Our understanding of which structural features of Rubisco control discrimination between the two gaseous substrates is rather meagre, and identification of determinants which influence CO2and O2substrate specificities is a prerequi-site for redirecting and modifying fluxes of glycolate-2-P and glycerate-3-P. Indeed, Rubisco is located at an ideal strategic position for control of photorespiration [8,11]. It is possible that the small subunit might influence both the enzymatic turnover and the discrimination of the two gaseous substrates [57,58•].
Despite a few impressive advances, it is fair to say that we still do not have a clear idea as to how any of these enzymes and transporters involved in photorespiratory cycle function at the molecular level in establishing the co-ordinated function of the C3- C2- and nitrogen-cycles for maximum efficiency. Likewise, an intriguing question is
how the co-ordinated control of a multitude of genes in a precise spatial and temporal program, can lead to the development of this exquisite photorespiratory cycle. It appears certain that the introduction of a genetic approach will complement the more classical methods used in the study and regulation of photorespiration with regard to the ultimate goal of engineering plants with superior growth characteristics and devising new herbicides.
Acknowledgements
This article is dedicated to the memory of Professor NE Tolbert, tireless champion of photorespiration.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest ••of outstanding interest
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