Photosynthesis Research 33: 147-162, 1992. © 1992 KluwerAcademic Publishers. Printed in the Netherlands. Minireview

Carbon dioxide assimilation in oxygenic and anoxygenic photosynthesis* Bob B. Buchanan Department of Plant Biology, University of California, Berkeley, CA 94720, USA Received 1 September 1991; accepted in revised form 12 March 1992

Key words:

thioredoxin, Co 2 assimilation, oxygenic photosynthesis, anoxygenic photosynthesis

Abstract

This article represents a summary of our contemporary understanding of carbon dioxide assimilation in photosynthesis, including both the oxygen-evolving (oxygenic) type characteristic of cyanobacteria, algae and higher plants, and the non-oxygen-evolving (anoxygenic) type characteristic of other bacteria. Mechanisms functional in the regulation of the reductive pentose phosphate cycle of oxygenic photosynthesis are emphasized, as is the reductive carboxylic acid cycle- the photosynthetic carbon pathway functional in anoxygenic green sulfur bacteria. Thioredoxins, an ubiquitous group of low molecular weight proteins with catalytically active thiols, are also described in some detail, notably their role in regulating the reductive pentose phosphate cycle of oxygenic photosynthesis and their potential use as markers to trace the evolutionary development of photosynthesis. Abbreviations: N A D P - G A P D H - NADP-glyceraldehyde 3-phosphate dehydrogenase; F B P a s e - fructose 1,6-bisphosphatase; F T R - ferredoxin-thioredoxin reductase; R u b i s c o - ribulose 1,5-bisphosphate carboxylase/oxygenase; S B P a s e - sedoheptulose 1,7-bisphosphatase; PRK - phosphoribulokinase; N A D P - M D H - NADP-malate dehydrogenase; C F 1 - A T P a s e - chloroplast coupling factor; G 6 P D H glucose 6-phosphate dehydrogenase

Introduction

Life on our planet obtains its substance and energy through the process of photosynthesis, a grand device by which photosynthetic organisms use the electromagnetic energy of sunlight to synthesize carbohydrates (CH20) and other cellular constituents from carbon dioxide and water. light

CO 2 + 2H20------~ (CH20) + 0 2 + H 2 0 Photosynthesis may be broadly divided into two phases: a light phase, in which the electro* Most of the references cited in this article are reviews. For references to specific material, readers should consult the appropriate review.

magnetic energy of sunlight is trapped and converted into ATP and NADPH, and a synthetic phase, in which the ATP and N A D P H generated by the light phase are used, in part, for biosynthetic carbon reduction. As described below, light also functions in the regulation of the synthetic or carbon reduction phase of photosynthesis and in related biochemical processes of chloroplasts (Buchanan 1980, 1991). In most plants, the major products of photosynthesis are starch (formed in chloroplasts), and sucrose (formed in the cytosol) (Cs6ke and Buchanan 1986). Both of these products are formed from photosynthetically generated dihydroxyacetone phosphate (DHAP) via pathways that in some respects are similar to the gluconeogenic pathway of animal cells. In the first case, D H A P is converted to hexose phos-

148 phates, which, in turn, are converted to starch within the chloroplast. In sucrose synthesis, D H A P (or a derivative) is transported to the cytosol and is there converted to sucrose. All oxygenic (oxygen evolving) organisms from the simplest prokaryotic cyanobacteria to the most complicated land plants have a common pathway for the reduction of CO 2 to sugar phosphates. This pathway is known as the reductive pentose phosphate (RPP), Calvin-Benson or C 3 cycle. Although the RPP cycle is the fundamental carboxylating mechanism, a number of plants have evolved adaptations in which CO 2 is first fixed by a supplementary pathway and then released in the cells in which the RPP cycle operates. One of these supplementary pathways, the C 4 pathway, involves special leaf anatomy and a division of biochemical labor between cell types. Plants endowed with this pathway, through greater efficiency, are able to flourish under conditions of high light intensity and elevated temperatures. A second supplementary pathway was found in species of the Crassulaceae and is called Crassulacean acid metabolism (CAM). These plants are often found in dry areas and fix CO 2 at night into C 4 acids. During the day, the leaves can close their stomata to conserve water while CO 2 released from the C 4 acids is converted to sugar phosphates by the RPP cycle using absorbed light energy. CO 2 fixation is also found in bacteria, both photosynthetic and non-photosynthetic. The purple sulfur and purple non-sulfur bacteria employ the RPP cycle, as do plants (Bassham and Buchanan 1982, Tabita 1988). The photosynthetic green sulfur bacteria, however, use ferredoxinlinked carboxylases in a pathway known as the reductive carboxylic acid cycle (Buchanan and Arnon 1990). The ferredoxin-linked carboxylases also function in CO 2 assimilation in diverse types of fermentative and methanogenic bacteria. Finally, in photosynthetic green non-sulfur bacteria, the path of carbon assimilation is unknown. In this article, we first describe the path of carbon in oxygenic photosynthesis (the reductive pentose phosphate cycle), concentrating on its mechanism of regulation, and then proceed to

the pathways in anoxygenic photosynthesis (the reductive pentose phosphate cycle, reductive carboxylic acid cycle, and the unknown pathway).

Oxygenic photosynthesis The crux of the pathway is the carboxylation of ribulose 1,5-bisphosphate to produce two molecules of 3-phosphoglycerate (Fig. 1). Sequentially, the next steps (the reductive phase of the cycle) are those in which ATP and NADPH, produced by the light reactions, are consumed in the reduction of 3-phosphoglycerate to glyceraldehyde 3-phosphate. To complete the cycle (the regeneration phase), intermediates formed from a portion of the glyceraldehyde 3-phosphate are utilized via a series of isomerizations, condensations and rearrangements that result in the conversion of glyceraldehyde 3-phosphate to pentose phosphate, eventually ribulose 5-phosphate. Phosphorylation of ribulose 5-phosphate by ATP regenerates ribulose-l,5-bisphosphate, thus completing the cycle. The portion of glyceraldehyde 3-phosphate not used to sustain the cycle constitutes net product and serves as a source of carbon for synthesis of chloroplast starch and for transport to the cytosol. The enzymes which catalyze steps in the cycle, identified in Table 1, 6ADP

Fig. 1. The reductive pentose phosphate cycle. Abbreviations: R u B P - ribulose-l,5-bisphosphate; P G A - phosphoglycerate; D i P G A - 1,3-diphosphoglycerate; G3P - glyceraldehyde 3-phosphate; D H A P - dihydroxyacetone phosphate; R u 5 P - ribulose 5-phosphate. The regulatory contribution of light is not included.

149 Table 1. Enzymes of the three phases of the reductive pentose phosphate cycle. Regulatory enzymes are shown in bold type

Phase of the cycle I. Carboxylation Carbon dioxide fixed into 3-phosphoglycerate II. Reduction 3-phosphoglycerate reduced to level of carbohydrate, net product formed III. Regeneration Initial carbon dioxide acceptor, ribulose 1,5-bisphosphate, regenerated

Constituent enzyme(s)

Ribulose 1,5-bisphosphate carboxylase / oxygenase

Phosphoglycerate kinase

Glyceraldehyde 3-phosphate dehydrogenase (NADP)

Triose phosphate isomerase Aldolase

Fructose 1,6-bisphosphatase Transketolase

Sedoheptulose 1,7-bisphosphatase Phosphopentoepimerase Phosphoriboisomerase

Phosphoribulokinase

are located in the soluble protein fraction of the chloroplasts, the stroma.

2

2v/l oc,,o Sll

Regulation of the reductive pentose phosphate cycle As is evident from Fig. 2, the principal and ultimate regulator of the 'dark' reactions (carbohydrate synthesis) is light (Buchanan 1980, 1991, Scheibe 1991). In fulfilling its regulatory role, light absorbed by chlorophyll and ultimately processed via ferredoxin is converted to regulatory signals that modulate selected enzymes. Such regulation, which takes place after the target enzymes are synthesized and assembled, is essential because enzymes for degrading carbohydrates coexist in chloroplasts with enzymes of carbohydrate synthesis. Selected biosynthetic enzymes are light-activated, whereas degradative enzymes are light-deactivated. In this way, chloroplasts minimize the concurrent functioning of enzymes or pathways that operate in opposing directions ('futile cycling') and thereby maximize the efficiency of temporally disparate metabolic processes. The regulatory function of light thus maintains 'enzyme order' by assuring that carbon dioxide assimilation takes place during the day, and carbohydrate degradation occurs primarily at night. Through the provision of triose phos-

- A0eo,s °62

Fig. 2. Contributions of the 'light' to the 'dark' reactions of photosynthesis in the assimilation of carbon dioxide.

phates (dihydroxyacetone phosphate) formed either from newly fixed carbon dioxide or from the breakdown of stored starch, chloroplasts are able to supply substrates for synthetic processes taking place in the cytosol. Primary among these processes is the synthesis of sucrose - a transport carbohydrate which in most plants satisfies day and night energy needs of non-photosynthetic (heterotrophic) tissues (Cs6ke and Buchanan 1986).

Sites of regulation The sensitivity of a metabolic pathway to regulation typically resides in a small number of the total steps in the pathway. Such regulatory steps usually have large, negative free energy changes and are thus essentially irreversible. The reactions that are substantially displaced from equilibrium in photosynthetic carbon assimila-

150 tion were proposed early as potential sites of metabolic regulation - ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), fructose 1,6bisphosphatase (FBPase), sedoheptulose 1,7bisphosphatase (SBPase) and phosphoribulokinase (PRK) (Bassham and Buchanan 1982). These sites were concluded to be important to the regulation of the pathway from changes in substrates and products in light/dark and dark/ light transition experiments. It would be expected that an increasing flux through a regulated step of the pathway would lead to depletion of the substrate for that step, and a decreasing flux would lead to a rise in concentration of the substrate. The kinetic analysis of such experiments is complicated by the cyclic nature of the pathway, since the production of a substrate for one reaction may be affected by the regulation of a subsequent step. Nevertheless, analyses confirm that the reactions catalyzed by FBPase, SBPase and PRK (regeneration phase) and Rubisco (carboxylation phase) are of greatest significance in controlling flux through the reductive pentose phosphate pathway. In contrast to the regeneration and carboxylation phases, the enzymes involved in the reduction phase (reduction of 3-phosphoglycerate to triose phosphate) together catalyze a freely reversible oxidation/reduction, the direction of which in vivo is largely determined by the levels of ATP and ADP and of NADPH and NADP. In the light, the high levels of ATP and NADPH drive the reactions in the direction of triose phosphate because of the sustained production of 3-phosphoglycerate and consumption of the newly formed triose phosphate. In steady-state photosynthesis this provides for a coordination of the activity of other parts of the cycle. Any component tending to increase the activity of PRK, for example, will cause consumption of ATP and production of ADP. The resulting deficiency of ATP, in turn, will slow the rate of 3-phosphoglycerate reduction, leading to decreased synthesis of ribulose 5-phosphate and bringing the cycle back into balance. It should be noted, however, that one enzyme of the reduction phase (NADP glyceraldehyde 3-phosphate dehydrogenase or N A D P - G A P D H ) is also regulated directly by light (see below).

Figure 3 summarizes some consequences that would result should key chloroplast enzymes become deregulated. It may be seen that the efficiency of starch buildup in the light and utilization in the dark would be seriously diminished. More specifically, deregulation of the enzymes indicated would lead to the following effects: (1) FBPase, wasted ATP, reduced ATP synthesis; (2) G6PDH, reduced net starch synthesis; (3) PRK, wasted ATP; (4) Rubisco, wasted ATP, phosphoglycolate formation; (5) SBPase, reduced ATP synthesis; and (6) NADP-GAPD, interference with Glu-6-P oxidation in the dark and triose phosphate transport to the cytosol. With reference to (3), the light deactivation (dark activation) of that G6PDH insures that the oxidative pentose phosphate cycle is active mainly in the dark when the products of the cycle, including NADPH, are not produced via photosynthesis.

The ferredoxin/thioredoxin system The ferredoxin/thioredoxin system, consisting of ferredoxin, FTR, and a thioredoxin, is a general mechanism of light-mediated enzyme regulation (Buchanan 1980, 1991, Scheibe 1991). Ferredoxin is a small protein, containing an ironsulfur group, that, among numerous other functions (Fig. 4) serves as the first soluble electron acceptor of photosynthetic electron transport. Ferredoxin was the first component of the ferredoxin/thioredoxin system to be identified (Buchanan 1991). Following elucidation of a requirement for FBPase activation, fractionation studies revealed that ferredoxin did not interact directly with the FBPase enzyme, but that a protein factor was required. Additional purification of this fraction revealed the presence of not one but two active chloroplast components that later were identified as a thioredoxin and FTR, the enzyme that reduces thioredoxin with ferredoxin. Another unexpected finding was made soon thereafter. On further purification the thioredoxin fraction was separated into two active components - thioredoxin f (named for its effectiveness in FBPase activation) and thioredoxin m (named for its effectiveness in NADP-malate

151 STARCH

Glu-6-P

FBP

Xu-5-P

G-3-P + DHAP

Ri-5-P

G-a-P

+

SBP

PG + PGA

~

NAD(P)H2~ NADPH2

],a-DiPGA

+ ATP

FBPase: Wasted ATP, reduced ATP synthesis G6PDH: Reduced net starch synthesis PRK: Wasted ATP

- -

-4-P

G-a-P

Rubisco: Wasted ATP, P-glycollate formation SBPase: Reduced ATP synthesis NADP-GAPDH: Interference with G6PDH reaction in dark and DHAP export

Fig. 3. Some consequences of deregulation of key chloroplast enzymes. Abbreviations not defined in text are: RuBP - ribulose 1,5-bisphosphate; P G - phosphoglycolate; PGA- 3-phosphoglycerate; Glu-6-P- glucose 6-phosphate; Fru-6-P- fructose 6-phosphate; FBP-fructose 1,6-bisphosphate; G-3-P-glyceraldehyde 3-phosphate; DHAP-dihydroxyacetone phosphate; 1,3DiPGA - 1,3-diphosphoglycerate; Xu-5-P - xylulose 5-phosphate; Ri-5-P - ribose 5-phosphate; SBP - sedoheptulose 1,7-bisphosphate; Sed-7-P- sedoheptulose 7-phosphate; Ery-4-P- erythrose 4-phosphate. NADP-GAPDH, FBPase, PRK, Rubisco and SBPase, which are denoted by the open bubble, are activated in the light and deactivated in the dark; G6PDH, denoted by the hatched bubble, is the opposite - it is activated in the dark and deactivated in the light. Deregulation means that these enzymes would be active continuously (in light and dark).

dehydrogenase or N A D P - M D H activation). These results d e m o n s t r a t e d that multiple forms of thioredoxin occur in the l e a f - a finding confirmed and extended with this and other tissues (see below). Thioredoxins are proteins, typically with a molecular weight of 12 k D a , that are now known to be widely distributed in the animal, plant and bacterial kingdoms. The initial studies with E. coli led to the identification and characterization of thioredoxin and the elucidation of its function as a hydrogen carrier in ribonucieotide reduction ( H o l m g r e n 1985). Thioredoxins have since been d e m o n s t r a t e d to fulfill a n u m b e r of cellular functions, including ones fundamental to regulation.

Thioredoxins undergo reversible reduction and oxidation through changes in a disulfide group (S-S---~ 2SH). In the ferredoxin/thioredoxin syst e m , a thioredoxin is reduced via an iron-sulfur enzyme, F T R , by ferredoxin, which itself is reduced by the electron transport system of illuminated chloroplast thylakoid m e m b r a n e s (Fig. 5). T h e enzymes so activated are oxidized, possibly by way of thioredoxin, and return to their inactive state in the dark (Fig. 6). [Readers wishing to read background material on thiols and their role in regulation, with an emphasis on chemistry, are referred to the review by Gilbert (1990).] As noted above, two different thioredoxins,

152 NAD (P)*

.oi

I

so; e

glutamate synthase

fatty acid desaturation e

N2

Light

Photosynthetic membranes

Electron donor

e

e

e

redactive carboxylic acid cycle

~1 FERREDOXJN

e

/

/

cyclic photophosphorylotion catalysis

/q,,, /l\ [

\

l

",. thioredoxin-reguloted enzymes

~r

noncycI|c phofophosphorylotlon catalysis

Fig. 4. Role of ferredoxin in metabolic processes.

f

Ferredoxikn

e-

2H+/

I 2Fe-2S-2e---> "~ 4Fe-4S FTRI Ferredoxin,k -S-S~2SH k 2H JThioredoxin I I-s-s--.~s~)- I form

\

Chlorophyll]

2

~

J

Fig, 5. Light activation of biosynthetic enzymes by the ferredoxin/thioredoxin system. T h e reduction of F T R requires two protons from the m e d i u m and two electrons from ferredoxin, a one-electron carrier. A s F T R appears to bind ferredoxin on an e q u i m o l a r basis, it is believed that there is a sequential transfer of one electron from ferredoxin in the reduction of the enzyme. T h e role of the iron-sulfur cluster of F F R is still an open question.

153 designated f and m, are a part of the ferredoxin/ thioredoxin system in chloroplasts. In the reduced state, thioredoxin f selectively activates enzymes of carbohydrate synthesis, including FBPase, SBPase, PRK and N A D P - G A D P H . Thioredoxin m preferentially regulates (deactivates in the light) glucose 6-phosphate dehydrogenase (G6PDH), a key enzyme of the oxidative pentose phosphate cycle, a route of carbohydrate degradation alternate to glycolysis. Thioredoxin m also functions in chloroplasts in activating N A D P - M D H - an enzyme especially important in C 4 plants, species forming C 4 acids such as malate as early photosynthetic products. N A D P MDH also occurs in chloroplasts of C 3 plants and there is believed to function as part of a lightdependent mechanism which transports excess reducing equivalents to the cytosol. Both thioredoxins are effective in activation of the 'coupling factor' (CFI-ATPase), an enzyme functional in photophosphorylation. The major groups of oxygenic photosynthetic organisms have been shown to utilize the ferredoxin/thioredoxin system in enzyme regulation. These include cyanobacteria, eukaryotic algae, C 4 and Crassulacean acid metabolism (CAM) plants in addition to C 3 plants for which the system was first described, i.e., species forming 3-phosphoglycerate as first stable carboxylation product. All eukaryotic oxygenic photosynthetic organisms examined contain both thioredoxins, f and m. Cyanobacteria, on the other hand, contain thioredoxin m, but seem to lack a typical thioredoxin f - a situation that is in accord with

the apparent difference in the evolutionary history of the two proteins (see below). The ferredoxin/thioredoxin system functions by changing the redox status of target enzymes. Biosynthetic enzymes are activated by a net transfer of reducing equivalents (hydrogen) from reduced thioredoxin to enzyme disulfide (S-S) groups, thereby yielding oxidized thioredoxin and reduced (SH) activated enzyme. Deactivation takes place through the oxidation (in the dark) of SH groups on reduced thioredoxin which in turn oxidizes the reduced (activated) enzyme. Enzymes of carbohydrate degradation regulated by this system show an opposite response, i.e., a deactivation on reduction and an activation on oxidation. During the past few years, striking progress has been made on the ferredoxin/thioredoxin system (Fig. 7). In one important development, the regulatory sites of several thioredoxin-linked enzymes have been identified (Fig. 8). FBPase and N A D P - M D H show a regulatory site that contains a cystine disulfide bridge near the center and amino terminus of the polypeptide chain, respectively. A situation similar to that of the FBPase holds for the regulatory (gamma) subunit of CF1-ATPase. In all three of these cases, the regulatory site is separate from the active site. In PRK, by contrast, the two sulfur groups of the regulatory site are a part of the active site. PRK is also unique in that the regulatory cyst(e)ines are separated by a large number of residues (thirty nine). The positioning of the regulatory site relative to the active site of chlo-

DEACTIVATION OF THIOREDOXIN-ACTIVATEDENZYMESIN THE D A R K BY 02

(i)

2 Enzyme (active) -SH HS-

+

(ii)

2 Thioredoxin

+

Sum:

2 Thioredoxin (oxidized) -S-S02

~

2 Enzyme (inactive) -S-S-

+

--I~-

2 Thioredoxin

+

2 H20

+

2 H20

(reduced)

(oxidized)

-SH HS-

-S-S-

2 Enzyrae (active) - S H HS-

Fig.

6.

+

02

--P"

2 Enzyme

2 Thioredoxin (reduced) -SH HS-

(inactive) -S-S-

Dark deactivationof biosyntheticenzymesby the ferredoxin/thioredoxinsystem.

154 Recent Progress on the Ferredoxin / thioredoxin System All components purified and characterized at protein level Genes cloned and sequenced: ferredoxin, thioredoxins m and f~ FrR (one subunit), several target enzymes Thioredoxin rn gene shown to be essential for growth of a cyanobacterium Physiological evidence for function with isolated intact chloroplasts (monobromobiraane, mBBr, probe) Identification of regulatory sites on target enzymes Initiation of phylogenetic studies

Fig. 7. Recent advances in the ferredoxin/thioredoxin system.

roplast FBPase, N A D P - M D H and PRK was confirmed in experiments with isolated intact chloroplasts. The nature of the regulatory sites of FBPase, N A D P - M D H and CF1-ATPase vs. PRK suggests a difference in the evolutionary history of these two groups of enzymes. In the future, it will be of interest to determine the phylogenetic relationship between these enzymes of chloroplasts and their counterparts from other sources, e.g., chloroplast FBPase, N A D P - M D H and CF1-ATPase and the corresponding enzymes of animals and microorganisms that lack a thioredoxin regulation site. It is noted that the reductire pentose phosphate cycle is not light regulated in anoxygenic photosynthetic organisms utilizing this pathway - an earlier conclusion supported by sequence information on the PRK from purple non-sulfur bacteria.

Enzyme

In yet another development, phylogenetic studies have been initiated on thioredoxins (Buchanan 1991). The similarity between the m-type thioredoxins of chloroplasts and the thioredoxins from a variety of bacteria has been extended in an analysis in which fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree (Fig. 9). When analyzed by a parsimony-based method, the bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria

(Chromatium, Rhodospirillum, Rhodopseudomonas) as well as E. coli and Corynebacterium, both heterotrophs; a second containing the photosynthetic green bacterium, Chlorobium, which was used to root the tree in Fig. 9; and a third containing cyanobacteria (Anacystis and Anabaena). These groupings are similar to those generated from earlier 16S ribosomal RNA analyses (Woese 1987). Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts ( f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but thioredoxin f grouped with animal thioredoxins. While the function of thioredoxin is not fully clear in animal cells, there is a growing body of evidence for a function in cell division independently of ribonucleotide reduction- the first reaction in which thioredoxin was found to participate (Holmgren 1985). The findings illustrate the potential of thiore-

Regulatory Site

Laboratory

FBPase

...Arg-Cys-Val-Val-Asn-Val-Cys-Gly... 174 179

Marcus, Dyer

NADP-MDH

...Glu-Cys-Phe-Gly-Val-Phe-Cys-Thr... lO 15

Gadal

PRK

...Gly-Cys-Gly- . . . -Ile-Cys-Leu... 16 55

Hartman

CFI-ATPase

...lle-Cys-Asp-Ile-Asn-Gly-Lys-Cys-Val... 199 205

Futai

Thioredoxin _f

...Trp-Cys-Gly-Pro-Cys-Lys-... 37 40

SchOrmann

Fig. 8. Regulatory sites of thioredoxin-linked chloroplast enzymes. A s indicated, the references for FBPase are Marcus et al. and Raines et al.; N A D P - M D H , Decottignies et al.; P R K , Porter et al.; C F 1 - A T P a s e , Miki et al.; and thioredoxin f.

155 34

CHLOROBIUM 26 SPINACH m , ~8 CHLAMYDOMONAS ~5 1 9 ANACYSTIS I Is I 8 A N A B A E N A 15

20

i3

~4 ~8

io 36

s2 30

CHROMATIUM E. COkl 22 RHODOSPIRrLLUM 25 RHODOPSEUDOMONAS

CORYNEBACTERIUM SPINACH f ~4 ~ HUMAN I " ~ ' L ~ . - RAB BIT I 14 CHICKEN

enzymes limit this regeneration. In a recent development, the sites on several of the enzymes regulated by thioredoxin have been identified, with interesting results both from the standpoint of protein structure and evolution. In the latter context, thioredoxins have emerged as a new evolutionary marker that is extending results obtained with other markers. Systems known to interact

1Q ~

Fig. 9. Phylogenetic tree for prokaryotic and eukaryotic thioredoxins based on amino acid sequences. The bar indicates a distance of 10 amino acid replacements.

doxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins. It will be of interest to determine the phylogenetic position of the third type of plant thioredoxin-extra chloroplastic thioredoxin h - a species that resembles animal thioredoxin structurally and in its intracellular localization endoplasmic reticulum, cytosol and mitochondria. Thioredoxin h is reduced with NADPH and a flavin enzyme, NADP-thioredoxin reductase, as first established for the thioredoxin system of bacterial and animal cells. In animals, as well as aerobic bacteria such as E. coli, thioredoxin is reduced by NADPH via a flavin enzyme, NADP-thioredoxin reductase. One possible physiological function of the plant NADP/thioredoxin system is the reduction of purothionin, a protein of seeds. Elucidation of other functions of this system poses an interesting question that is currently being addressed (see below). To sum up, the evidence is consistent with the view that the ferredoxin/thioredoxin system functions in diverse types of oxygenic photosynthetic organisms as a light dependent mechanism for the regulation of both biosynthetic and degradatory enzymes. Aside from N A D P GAPDH, the enzymes of the reductive pentose phosphate cycle controlled by this system (FBPase, SBPase and PRK) act to regenerate the carbon dioxide acceptor, ribulose-l,5-bisphosphate, from newly formed 3-phosphoglycerate. It seems likely that thioredoxin-linked

Biochemical .processes are generally regulated not by one, but by several interacting systems of regulation. From early work, it was concluded that the ferredoxin/thioredoxin system acts jointly with other light-mediated systems, i.e., light-driven shifts in pH, divalent cations and in the concentration of metabolite effectors (Buchanan 1980). Since those studies, results from a number of laboratories support such a coordinate function of the different regulatory systems (Buchanan 1991, Scheibe 1991). Noteworthy among the metabolite effector studies are the demonstration of the inhibition of thioredoxin-linked N A D P MDH activation by NADP, the inhibition of activation of PRK by compounds such as 6phosphogluconate and the enhancement of thioredoxin-linked FBPase and SBPase activation by substrate (sugar bisphosphate), and divalent cations (Ca ++ and Mg++). Agents that alter hydrophobic interactions also enhance thioredoxin effects. In short, it appears that the ferredoxin/thioredoxin system functions jointly with mechanisms promoting light-dependent shifts in ions and metabolites in the regulation of a number of chloroplast enzymes. Rubisco linked systems Rubisco is one regulatory enzyme of the carbon cycle that utilizes mechanisms of regulation other than the ferredoxin/thioredoxin system. These include (1) activation by formation of a complex 2+ with carbon dioxide and Mg ; (2) activation by interaction with a novel enzyme, rubisco activase in a light-dependent reaction that appears to depend on the ion gradient generated by chloroplasts; and (3) inhibition by 2-carboxyarabinitol 1-phosphate, a compound whose concentration

156 changes in the light and dark. The reader is referred to general articles for a discussion of our current understanding of rubisco regulation (Campbell and Ogren 1990, Gutteridge 1990).

Possible future developments The elucidation of the role of thioredoxin in targeting specific sites on chloroplast enzymes opens the door to a new technology (Buchanan 1991). By using protein engineering techniques, it is now possible to alter the thiol redox properties of proteins, including the capacity for regulation. A recent study on the in vitro mutagenesis of E. coli thioredoxin is a case in point. Replacement of aspartate by aspargine at position 61 significantly increased the ability of this thioredoxin to activate FBPase when reduced either photochemically be ferredoxin and FTR or chemically by DTT. Also through sitedirected mutagenesis, it was feasible to enhance the stability of PRK through modification of a thioredoxin-linked cysteine group. Finally, in another study, it was found that substitution of cysteine for threonine at positions 21 and 142 of bacteriophage T4 lysosome made possible the reversible redox regulation of the enzyme by thiol reagents. These examples illustrate that the thiol redox properties of enzymes can be successfully modified and raise the possibility that engineered alterations in regulation will find application in controlling enzyme activities, eventually in industrial processes. Thiol changes of the type found with chloroplasts also appear to occur during the maturation and germination of seeds, but here the changes are linked to development rather than photosynthesis (Kobrehel et al. 1992). Quite recent evidence suggests that thioredoxin (h type) plays a regulatory role in seed germination through the reduction of disulfide groups of storage proteins as well as specific enzymes. Such reductive changes appear to enhance the solubilization and mobilization of storage proteins and the activation of 'dormant' enzymes. Interestingly, the redox changes triggered by thioredoxin in seeds appear to be relevant to industrial processes. The results suggest that the preparation of certain food products derived from seeds (e.g., bread prepared from poor quality flour) can be

improved by the modification of components of the NADP/thioredoxin system during processing (K. Kobrehel, J.H. Wong, B.C. Yee and B.B. Buchanan, unpublished findings). Future applications also exist with leaves. As the regulatory sites of chloroplast enzymes targeted by thioredoxin are not found in the corresponding proteins of animal or microbial cells, it may be feasible to design new thiolspecific herbicides. Such herbicides could target an enzyme equally important in all types of plants (e.g., chloroplast FBPase) or an enzyme more abundant in a particular plant group, such as N A D P - M D H in C 4 species. Finally, it is anticipated that new thioredoxinlinked enzymes will be identified in oxygenic photosynthetic cells. The ferredoxin/thioredoxin system has been reported to function in the regulation of enzymes outside the immediate arena of carbon dioxide assimilation, i.e., in the metabolism of nitrogen, sulfur and glycerol, a lipid precursor. It is likely that future work will extend the function of thioredoxin to the regulation of other cell processes. Such processes may include ones unique to fermentative bacteriaorganisms known to contain a novel thioredoxin system in which ferredoxin supplies reducing equivalents via a flavin enzyme. To sum up, the mechanism of carbon dioxide assimilation by the reductive pentose phosphate cycle has been known for almost four decades. During this time, it has been established that light functions not only to fulfill ATP and N A D P H requirements, but also to regulate selected enzymes of the cycle and of processes influencing its operation. Historically, the reactions catalyzed by these enzymes have been known as the 'dark' reactions - a name that does not reflect their dependency on light for regulation. In oxygen-evolving systems (chloroplasts and cyanobacteria), light absorbed by chlorophyll is converted to several different regulatory signals that alter the activities of enzymes of the 'dark' reactions- changes in pH, divalent cations, metabolite effectors, and sulfhydryl groups. Collectively these signals 'inform' selected enzymes that the light is on and that their activities should be altered accordingly. In the case of sulfhydryl changes, the light signal is carried from chlorophyll containing thylakoid

157 membranes via ferredoxin to thioredoxins, which, through redox changes in their own sulfhydryl groups, bring about reversible changes in the sulfhydryl status of target enzymes. Such changes alter the activities of key enzymes and direct major biosynthetic and degradatory pathways in the appropriate direction. With certain enzymes, the light-produced alkalization of the stroma and the increase in the concentration of cations and selected metabolite effectors enhance the sulfhydryl changes. By linking these regulatory changes to light, the cell is in command of its biosynthetic and degradatory capabilities at all times and can direct available resources to increase growth and survival under a wide range of environmental conditions. It is significant that photosynthetic bacteria (anaerobic photosynthetic organisms that lack the ability to evolve oxygen) seemingly did not evolve a mechanism to regulate metabolic processes in this manner, possibly due to the absence of a suitable oxidant during growth. Current evidence suggests that it may be possible to alter the response of enzymes of this type to thioredoxin through protein engineering. Such a change is but one of a number of potential future applications stemming from thioredoxin regulation research.

Anoxygenic photosynthesis

The reductive pentose phosphate cycle Photosynthetic bacteria assimilate CO 2 via basically different pathways: (1) the reductive pentose phosphate cycle (C 3 cycle or Calvin cycle) and its associated reactions as in oxygenic photosynthetic organisms (Bassham and Buchanan 1982, Tabita 1988); and (2) ferredoxin-linked reactions of the reductive carboxylic acid cycle (reverse citric acid cycle) that are unique to prokaryotic cells (Buchanan and Arnon 1990). Each of these routes is discussed later in relation to bacterial photosynthesis. General aspects of the reductive pentose phosphate cycle are more fully discussed earlier. In photosynthetic bacteria in which the reductive pentose cycle is predominant, an important carboxylase, i.e., one functional in addition to

RuBP carboxylase, is PEP carboxylase. In these organisms, PEP carboxylase appears to function in the formation of C 4 acids, analogous to its role in C 4 plants. These bacteria also possess ferredoxin-linked pyruvate synthase and, in some cases, other ferredoxin-linked carboxylases described below.

The reverse citric acid cycle The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It occupies a central position in the metabolism of most biochemically significant compounds. It liberates CO 2 and generates N A D H whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO 2 assimilation. In 1966, a cyclic pathway for CO 2 assimilation was described that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO 2 by a pathway different from Calvin's reductive pentose phosphate cycle and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO 2 assimilation pathway in Chlorobium thiosulfatophilum (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grown anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO 2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the Calvin cycle was an important property shared by all autotrophic species, but also some of its experimental underpinnings were challenged. Our aim here is to summarize (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO 2 assimi-

158 lation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

Origin of the concept of the new cycle The concept of the reductive carboxylic acid cycle had its origin in the discovery of ferredoxin-linked reductive carboxylation reactions (Buchanan and Arnon 1990). In the reductive pentose phosphate cycle the reductant is N A D P H (Em, 7 = -320 mV). When it was found that ferredoxin and not N A D P H is the first stable carrier of photosynthetically generated reducing power and that its midpoint potential is about equal to that of molecular hydrogen (Era. 7 = - 4 2 0 m V ) the possibility arose that reduced ferredoxin may serve directly as a reductant in reductive carboxylations instead of participating indirectly by way of pyridine nucleotides with an attendant 100 mV loss in reducing potential. The first substantiation of this possibility was the discovery that reduced ferredoxin could drive a net reductive synthesis of pyruvate from acetylCoA and CO 2 (Eq. (1)). Acetyl-CoA

+ C O 2 -t- 2Fdre d +

Pyruvate + CoA + 2Fdo~

2H+ (1)

The reaction was in essence a reversal of the oxidative decarboxylation of pyruvate through which acetyl-CoA is supplied to the Krebs cycle. The enzyme catalyzing the new reaction was detected in cell-free extracts of the fermentative anaerobe, Clostridium pasteurianum. The enzyme catalyzing the reaction, named pyruvate synthase (now also known as pyruvate ferredoxin oxidoreductase), was later purified to homogeneity from a related organism, Clostridium acidi-urici. After initial experiments with ferredoxindependent carboxylations in fermentative bacteria that live in the soil independently of light, such carboxylations were also found in phot~ synthetic bacteria that live autotrophically in the light by means of an anaerobic (anoxygenic) type of photosynthesis. Here reducing equivalents are supplied not by water but by reductants such as

hydrogen sulfide or thiosulfate. Pyruvate synthase was found in cell-free preparations from each of the three then known groups of photosynthetic bacteria: the already discussed Chlorobium representing the green sulfur group; Chromatium strain D (now called Chromatium vinosum) representing the purple sulfur group; and Rhodospirillum rubrum representing the purple non-sulfur group. Furthermore, the activity of pyruvate synthase explained previously puzzling 14C-labelling results in bacterial photosynthesis obtained with whole cells. The next important development that led directly to the formulation of the carboxylic acid cycle was the discovery of a-ketoglutarate synthase (Eq. (2) in Chlorobium and later (with low activity) in R. rubrum. Succinyl-CoA

+

CO 2 q- 2Fdre d + 2H +

~-Ketoglutarate + CoA + 2Fdox ) .

(2)

-ketoglutarate synthase aroused special interest because it exemplified the use of the reducing power of ferredoxin to reverse the step in the Krebs cycle hitherto considered to be irreversible, i.e., the decarboxylation of ~-ketoglutarate to succinyl-CoA and CO 2. Its action was similar to that of pyruvate synthase (Eq. (1)) in that both enzymes catalyzed reductive ferredoxindependent carboxylations of an acyl-CoA derivative to form the corresponding ~-keto acid (Eq. (3)). R - CO - S - CoA + CO 2 + 2Fdre d + 2H ÷ R - CO - C O O H + CoA - SH + 2Fdox (3) This similarity raised the question whether pyruvate synthase and ~-ketoglutarate synthase were one or two distinct enzymes. Later studies with purified preparations showed that o~-ketoglutarate synthase is indeed distinct from pyruvate synthase with respect to molecular mass and chromatographic properties.

The reductive carboxylic acid cycle The new ferredoxin-dependent enzymes, pyruvate synthase and a-ketoglutarate synthase, formed the cornerstones of the reductive carbox-

159

C5

a.

c~~

C3

C5

B.

Fig. 10. (A) Schematic overview of the net synthesis of a C 2 product via the reductive carboxylic acid cycle. (B) Schematic overview of the net synthesis of a C 4 product via the reductive carboxylic acid cycle.

ylic acid cycle (Buchanan and Bassham 1982, Buchanan and Arnon 1990). Here, the overall chemical change is the net synthesis from CO 2 (i) of acetyl-CoA starting with oxalacetate, or (ii) of oxalacetate starting with acetyl-CoA. The continuing cyclic reaction sequences are the formations of mono-, di- and tricarboxylic acids. Beginning with oxalacetate, two sequential additions of CO 2 (C4--~ C 5--~ C6) give citrate (the C's refer to oxalacetate, a-ketoglutarate, isocitrate/citrate). Cleavage by citrate lyase yields acetyl-CoA as the net product and regenerates oxalacetate which gives rise to a four-carbon CO 2 acceptor (Figs. 10A and B). Beginning with acetyl-CoA, the cycle operates by four sequential additions of CO 2 (C 2 --~ C 3 -~ C 4~ C s ~ C6) ATP CoA

J~

/ ~

/

/

/

producing citrate (the Cs refer to acetate, pyruvate, oxalacetate, ~-ketoglutarate, isocitrate/ citrate). The citrate is cleaved by citrate lyase, yielding oxalacetate as product and regenerating acetyl-CoA as CO 2 acceptor (Fig. 11). The reductive carboxylic acid cycle is well suited to provide directly or indirectly building blocks for all other cellular constituents. For example, acetyl-CoA may be used directly for lipid synthesis or be converted to pyruvate which in turn may give rise, with or without additional carboxylations by the cycle, to amino acids (e.g., alanine, aspartate, glutamate) or to sugar phosphates (glucose, fructose) via gluconeogenesis. Participating in the latter pathway in photosynthetic bacteria is pyruvate-Pi-dikinase, an enzyme that also functions in certain higher plants. Evidence for the operation of the reductive carboxylic acid cycle as a full-fledged photosynthetic path for CO 2 assimilation operating independently of the reductive pentose phosphate cycle was assembled most completely for Chlorobium (Buchanan and Arnon 1990). The original evidence for the cycle included (i) demonstration in cell-free preparations, by measurements of enzymatic activity and 14C tracer distribution, of all the enzymes needed to catalyze the reactions of the cycle proper and of the associated reactions needed for biosynthesis, (ii)

CITRATE,~.,~,.,..

cls-AOO~ATE ,SOC,T~TE~-..~

succ,YL-CoA PHOSPHOENOL~ PYRUVATE~ .

SUC~NATE

Z~.~

Fig. 11. The reductive carboxylic acid cycle.

160 identification of the expected metabolic products in short-exposure 14CO2 experiments with growing cells, and, as discussed below, (iii) evidence that ChIorobium lacks the key enzymes of the pentose cycle for photosynthetic CO 2 assimilation. Moreover, it had just been demonstrated that reduced ferredoxin which drives the cycle is photochemically generated by Chlorobium. Evidence for the cycle was soon materially strengthened by independent inhibitor and 14Clabeling studies from the laboratory of J.G. Ormerod and R. Sire~g with washed suspensions of Chlorobium cells. This support, however, did not avert the ensuing controversy. The controversy The reductive pentose phosphate cycle is uniquely characterized by the operation of two key enzymes: ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBP, formerly RuDP carboxylase, or rubisco) and phosphoribulokinase (PRK). In fact, advocates of this cycle as the exclusive pathway for autotrophic CO 2 assimilation proposed that the acquisition of RuBP carboxylase and phosphoribulokinase may have coincided with emergence of autotrophic life. CO 2 assimilation in Chlorobium, a strict autotroph, directly contradicted this generalization. One way to resolve this contradiction was to test for the presence in Chlorobium of the two marker enzymes of the pentose cycle, rubisco and PRK. Their presence would reaffirm the universality of the pentose cycle for autotrophic CO 2 assimilation and relegate the new carboxylic acid cycle to an 'ancillary role'. Conversely, the absence of rubisco and PRK in Chlorobium would indicate that an alternate pathway for CO 2 assimilation such as the carboxylic acid cycle must indeed operate to account for the strictly autotrophic growth of this organism. At first it appeared that despite our doubts to the contrary, rubisco may be present in Chlorobium. Other investigators isolated the enzyme from Chlorobium and described certain of its properties. These results, still lacking confirmation, were found in other studies to be due to errors introduced by contaminating microorganisms. Furthermore, extensive attempts in several laboratories yielded no evidence for the activity

of either rubisco or PRK in Chlorobium. Similar results were obtained by other investigators who used both enzymatic and molecular genetic approaches. Finally, despite continuing reservations, mass ratio 13C analyses and 14C labelling studies with growing Chlorobium cells rules out a significant role for the pentose cycle in this organism and produced strong evidence for the operation of the carboxylic acid cycle, as the main pathway of CO 2 assimilation in Chlorobium. Questions were also raised about the presence in Chlorobium of some of the key enzymes required for the carboxylic acid cycle, specifically, the ATP-linked citrate lyase. Some early work contradicted our initial findings and suggested its absence but later investigations showed that activity of this enzyme in Chlorobium preparations was greatly stimulated by dithiothreitol. When proper precautions were exercised, as observed in the laboratory of E. Kondrateva, ATPlinked citrate lyase was consistently found in Chlorobium preparations, also in laboratories that had earlier reported negative findings. In sum, the original documentation of the cycle has been validated and greatly enlarged by extensive studies in other laboratories. The operation of the cycle is now generally accepted and has ceased to be a subject of controversy (Tabita 1988). An interesting question remaining is to what extent the reductive carboxylic acid cycle operatives in the oxidative direction in Chlorobium cells maintained in the dark. A n unknown pathway As described above, carbon dioxide is assimilated in anoxygenic photosynthesis either via the reductive pentose phosphate cycle (photosynthetic purple sulfur or non-sulfur bacteria) or the reductive carboxylic acid cycle (photosynthetic green sulfur and purple non-sulfur bacteria). The assimilation path operative in another major group of these organisms (photosynthetic green non-sulfur bacteria) is a mystery (Ormerod and Sirev~g 1983, Holo and Grace 1987). The member of this group studied, Chloroflexus auranticus, lacks the enzymes diagnostic of both of the known carbon pathways. The evidence suggests that a yet-to-be discovered carbon pathway is in

161 the green nonsulfur bacteria - organisms that appear to have deep evolutionary roots (Woese 1987). Elucidation of the carbon pathway functional in Chloroflexus is one of the interesting questions in this field.

Evolutionary implications So long as the reductive pentose phosphate cycle was thought to be the only mechanism for autotrophic CO 2 assimilation, the emergence of autotrophic life would have to coincide with the emergence of that cycle. The recognition of the reductive carboxylic acid cycle in a photoautrophic organism like Chlorobium suggests another scenario that seems more probable. On an evolutionary scale, ferredoxin is an ancient, low potential (equivalent to molecular hydrogen) metalloprotein electron carrier, that probably functioned in the earliest living organisms. These, it is generally agreed, were anaerobes. Then as now, ferredoxins were key participants in oxidoreductions that involve strong reducing potentials. As discussed above, clostridia and other anaerobic bacteria, both photosynthetic and non-photosynthetic, possess ferredoxin-dependent reductive carboxylation enzymes, pyruvate synthase and a-ketoglutarate synthase, that are the cornerstones of the reductive carboxylic acid cycle uncovered in Chlo-

robium. Early work buttressed the concept that as molecular hydrogen vanished from the primitive atmosphere, ancient phototrophs became dependent on the photosynthetic apparatus for the generation of strong reductants like reduced ferredoxin from such electron donors as thiosulfate. Support for this view came from similarities found among Chlorobium, Chromatium and clostridial ferredoxins. From this point of view, the reductive carboxylic acid cycle would have evolved in an organism like Chlorobium that already had ferredoxin and a complement of enzymes capable of catalyzing ferredoxin-dependent carboxylations. Hence, the reductive carboxylic acid cycle rather than the reductive pentose phosphate cycle, would have been the more likely pathway for CO 2 assimilation in primitive photosynthesis typified by Chlorobium. The reductive pentose

phosphate cycle would be a later development that has probably evolved by a reversal of the oxidative pentose cycle and the acquisition of the rubisco and PRK enzymes. Is there an evolutionary connection between ancient pathways of CO 2 assimilation characteristic of obligate anaerobes and the Krebs cycle that is the hallmark of aerobic metabolism? Krebs himself seemed to think so (see Buchanan and Arnon 1990). Oxygen appeared in the atmosphere as a result of oxygenic photosynthesis, which, on the scale of evolution, had evolved later than the anoxygenic photosynthesis typified by Chlorobium. From this perspective, the carboxylic acid cycle may indeed have an ancestral relation to the Krebs cycle. In the course of evolution, the biosynthesis of acetate (acetyl-CoA) from CO 2 by the carboxylic acid cycle may have been transformed into the citric acid cycle that degrades acetate into CO 2. Such transformation would be in harmony with the use of mechanisms that had evolved in earlier epochs in connection with other functions.

Note added in proof Evidence published while this article was in press indicates that the reductive carboxylic acid cycle is not confined to photosynthetic anaerobes. Chen and Gibbs reported the presence of pyruvate and a-ketoglutarate synthases, two key enzymes of the cycle, in a phosphoribulokinase deficient mutant of the eukaryotic green alga, Chlamydomonas reinhardtii (1992 Plant Physiol 98: 535-530). This finding is consistent with the view that the reductive carboxylic acid cycle functions in oxygenic photosynthesis.

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