Respiratory Processes in Anoxygenic and Oxygenic Phototrophs

Respiratory Processes in Anoxygenic and Oxygenic Phototrophs Roberto Borghese and Davide Zannoni University of Bologna, Bologna, Italy

Phototrophic microrganisms include “anoxygenic phototrophs,” which are bacteria capable of growing photosynthetically with no oxygen generation, and Cyanobacteria which are “oxygenic phototrophs” because their photosynthetic apparatus generates oxygen. Several genera of anoxygenic phototrophs are capable of obtaining energy also from aerobic and anaerobic respiration in darkness; conversely, only a few filamentous cyanobacteria can grow in the dark on glucose or other sugars using the organic material as both carbon and energy source. The latter observation suggests that besides the bioenergetic aspect, respiration in cyanobacteria plays other roles such as to control the redox balance or to act as a scavenger for O2 during nitrogen fixation. Facultative phototrophs (capable of both respiration and photosynthesis) contain a photosynthetic apparatus whose synthesis is repressed by oxygen; an exception to this rule is the group of aerobic-anoxygenic phototrophs, mainly marine microorganisms, requiring the presence of oxygen to synthesize their photosynthetic apparatus.

electron acceptors. Further, some strains of the species Rhodopseudomonas (Rps.) palustris, Roseobacter (Rsb.) denitrificans and Rba. sphaeroides can reduce 2 nitrate (NO2 3 ) into dinitrogen (N2) via nitrite (NO2 ), and, in some cases, also nitric oxide (NO) and nitrous oxide (N2O). These energy generating processes are catalyzed by oxido-reduction protein complexes forming composite electron transport chains. Apparently not all the above summarized metabolic options are activated or can be available simultaneously in a single species; however, cells of Rba. capsulatus or Rba. sphaeroides grown photosynthetically in the presence of low oxygen tension (, 1%) contain both photosynthetic and respiratory apparatuses.

ELECTRON TRANSPORT CHAINS

Facultative phototrophs are probably the most metabolically flexible organisms of the microbial world. Species such as Rhodobacter (Rba.) capsulatus and Rba. sphaeroides can grow by aerobic respiration and photosynthesis using either organic or inorganic substrates but also by anaerobic respiration with trimethylamineN-oxide (TMAO) or dimethyl sulfoxide (DMSO) as

Respiration in facultative phototrophs involves numerous redox carriers, namely: (1) transmembrane protein complexes such as NADH- and succinate-quinone oxidoreductases (NQR and SQR, respectively), cytochrome (cyt) bc1 or hydroquinone-cytochrome c oxidoreductase (QCR), quinol oxidase(s) (QOX), and cyt cbb3 and/or aa3 oxidases (COX); (2) electron and/or proton carriers such as ubiquinones (UQ), cyt c2, HiPIP, and cyt cy; (3) enzymes of the periplasmic space such as 2 NO2 3 , NO2 , N2O, and DMSO reductases. With O2 as final electron acceptor, the NQR, QCR, and COX enzymes constitute three main coupling sites where the potential energy between the initial donor and the final acceptor molecules is released in small steps, that are controlled by the differences between the redox midpoint potentials (Em) of the redox couples involved, and efficiently coupled to the generation of an electrochemical proton gradient (DmHþ). Photosynthesis converts the radiant energy into chemical energy at the level of the photochemical reaction center (RC). This transmembrane protein complex generates a charge separation that is followed by a cyclic electron transfer involving quinone molecules (UQ-10), cyt bc1 complex, and soluble cyt c2 in addition to the membrane-bound cyt cy, in the case of Rba. capsulatus. Under dark aerobic

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Anoxygenic Phototrophs On a phylogenetic basis (16S rRNA analyses) phototrophic bacteria and their relatives are grouped in a class, the Proteobacteria, which is formed by several subclasses, named a, b, g, d, and 1. Facultative photosynthetic bacteria belong to a and b subclasses, e.g., genera Rhodobacter, Rhodospirillum, Rhodocyclus, Rubrivivax, and Erytrobacter.

METABOLIC ASPECTS OF FACULTATIVE ANOXYGENIC BACTERIA

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RESPIRATORY PROCESSES IN ANOXYGENIC AND OXYGENIC PHOTOTROPHS

Nitrate reductase DMSO reductase

??? QOX2

O2

QOX1

NADH deh

C2 Succinate deh

bc1 Cyy

UQ/UQ2

cbb3

O2

aa3

Hydrogenase C22 C

Sulfide-UQ reductase

hn

Nitric oxide reductase Nitrous oxide reductase

RC

FIGURE 1 Block scheme illustrating the complex network of electron transport chains in Rba. sphaeroides. Black arrows indicate the influx of reducing equivalents and the efflux of electrons leading to the membrane-bound oxidases. Dashed black arrows indicate output of electrons involved in anaerobic respiration. Gray-colored redox components and arrows are those specifically involved in photocyclic electron transfer while those shaded off are shared by photosynthesis and respiration. RC, photochemical reaction center; hn, radiant energy; C2, soluble cyt c2; Cy , membrane-attached cyt cy; QOX1, functional ubiquinol oxidase (qxtAB operon); QOX2, not functional ubiquinol oxidase (qoxBA operon).

conditions, facultative phototrophs use a respiratory chain, closely related to that present in mitochondria. The last step of O2 reduction into H2O is catalyzed by two oxidases (not always present in a single species): a cyt c oxidase (of cbb3 and/or aa3 type), inhibited by mM cyanide, and a quinol oxidase (of bo type), inhibited by mM cyanide, branching the electron flow at the level of the UQ-10 pool. Figure 1 shows a block scheme of the different electron transport pathways operating in Rhodobacter sphaeroides.

membrane; thus, the different redox chains are not necessarily in thermodynamic equilibrium. For example, two functional pools of soluble cyt c2 exist in Rba. sphaeroides and Rba. capsulatus. Further, the membrane-bound cyt cy is a direct electron donor to the cbb3-type oxidase of Rba. capsulatus or to the cbb3/aa3 type oxidases of Rba. sphaeroides. Consequently, the redox chain containing cyt cy must be organized in supramolecular complexes and operate independently of the redox state of the other respiratory pathways.

INTERACTION BETWEEN THE DIVERSE ELECTRON TRANSPORT CHAINS

AND

The interaction between the different bioenergetic chains of photosynthetic bacteria occurs by two nonexclusive mechanisms, namely: (1) an indirect mechanism, exerted by the proton motive force (PMF) as a “back pressure” on the rate of electron transport catalyzed by redox complexes, and (2) a direct mechanism of interaction between electron carriers, e.g., UQ-10 or cyt c2, that are engaged in multiple bioenergetic processes. In this way, the activity of a given bioenergetic chain would affect the redox state of the components in common and, consequently, the functioning of the other chains. Respiratory and photosynthetic apparatuses are localized in different parts of the internal bacterial membrane (CM); this implies the diffusion of some electron carriers such as UQ, in the lipid phase, or cyt c2, in the periplasmic space. However, the diffusion of these elements does not occur on the entire internal

Oxygen is the key factor in the coordinated regulation of respiratory and photosynthetic activities since it participates, directly or indirectly, in determining the levels of expression of all components involved in these processes (Figure 2). The main regulatory element in Rba. capsulatus is the RegA/RegB couple (PrrA/PrrB in Rba. sphaeroides), which is able to sense the change in O2 partial pressure in the environment, and to transform this information into a regulatory response. Within this couple, RegA is the effector component while RegB is the sensor partner. This sensor-effector couple functions according to a widespread regulatory model in which the sensor protein (RegB) detects changes in the environment, in this case variations in O2 tension, and modifies the effector protein (RegA) by phosphorylating it. Depending on its phosphorylated or dephosphorylated status, the effector can regulate the expression of

GENETIC REGULATION OF RESPIRATION PHOTOSYNTHESIS IN FACULTATIVE PHOTOTROPHS

RESPIRATORY PROCESSES IN ANOXYGENIC AND OXYGENIC PHOTOTROPHS

RegA RC

cy DH

Q pool

cbb3

bc1

O2

c2 QOX

AerR

O2

HvrA

FnrL

FIGURE 2 Genetic regulatory network of respiratory and photosynthetic activity in Rhodobacter capsulatus. Black and gray arrows indicate regulation under aerobic and anaerobic conditions, respectively. Straight lines are for positive regulation (induction), dotted lines are for negative regulation (repression). Genetic regulators are written in black while respiratory/photosynthetic components, connected by thin arrows, symbolising electron flow, are in dark gray. DH, NADH dehydrogenase; RC, photosynthetic reaction center; Q pool, ubiquinone-10 pool; bc1, transmembrane cyt bc1 complex; cy, membraneanchored cyt cy; c2, periplasmic (soluble) cyt c2; cbb3, membranebound cyt c oxidase; QOX, membrane-bound quinol oxidase. See text for further details.

a number of genes. RegA/RegB is a general aerobic/ anaerobic regulatory couple that influences the expression of many processes in addition to respiration and photosynthesis: N2 fixation, CO2 fixation, and H2-ase activity. The electron transport chain (ETC) genes that have been shown to be regulated by RegA/ RegB are the ones coding for COX and QOX, which are specific for the respiratory chain, and those coding for the bc1 complex, cyt c2 and cyt cy which participate in both respiration and photosynthesis (Figure 2). RegA/ RegB also regulate the level of the RC. Other regulatory elements that participate in the regulation of respiratory ETC components are AerR, that operates in the presence, as well as in the absence, of O2; HvrA and FnrL are anaerobic regulators only (Figure 2). Although the interaction of all the aforementioned regulatory elements is quite complex, it allows the fine tuning and controlled interplay of respiratory and photosynthetic activities.

Oxygenic Phototrophs Cyanobacteria are capable of oxygenic photosynthesis, differing in that from anoxygenic phototrophs. Cyanobacteria form one of the major phila of Bacteria and they were most likely the first oxygen-evolving organisms on Earth changing the atmosphere from anoxic to oxic. Oxygenic phototrophs are grouped into several

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morphological groups; however, most of the available biochemical and genetic data concern mainly unicellular genera such as Synechococcus and Synechocystis. Respiration is by definition a membrane-bound process; in this respect, cyanobacteria contain three different types of membranes: (1) the outer membrane, typical of Gram negatives, which has no specific role in respiration; (2) the cytoplasmic membrane (CM); and (3) the intracytoplasmic membranes (ICMs) or thylakoids. Both CM and ICM contain respiratory redox complexes; electron microscopy also indicates that CM and ICM might be connected, at least in Synechococcus sp. strain PCC 6301, although a correct picture of the membrane arrangement in vivo is lacking at present.

ELECTRON TRANSPORT PATHWAYS IN OXYGENIC PHOTOTROPHS Respiratory and photosynthetic electron transports are intimately connected in two distinct bioenergetically active membranes, ICM and CM. All photosynthetic electron transport is located in the ICM, where photosynthesis and respiration share components (Figure 3). In addition, ample experimental evidences indicate the presence of respiratory chain(s) in the CM. Cyanobacterial respiratory terminal oxidases (RTOs) have no direct function in photosynthesis and therefore can be considered the key enzymes of respiration. All cyanobacteria (investigated so far) contain several respiratory branches ending in different RTOs but their actual location in the membrane cell (ICM, CM, or both) is far from being assessed. The best-characterized species is Synechocystis sp. strain PC 6803, for which the complete genomic sequence is available. Three sets of genes for RTOs were found, the wellcharacterized aa3-type cyt c oxidase (COX, encoded by coxBAC), a related set of genes also belonging to the heme-copper oxidase superfamily, termed alternate RTO (ARTO; encoded by ctaCII-ctaDIIEII), and two genes (cydAB) encoding a putative cyt bd-type quinol oxidase (Cyd). The CM forms the inner boundary of the periplasmic space and is known to contain proteins typically associated with respiratory electron transport, such as NAD(P)H dehydrogenase, cyt b6/f (homologous of the bc1 complex), and terminal oxidases (presumably, ARTO). Two types of NAD(P)H dehydrogenase have been found. One is a NADPH-type I dehydrogenase (NDH-1), that is encoded by ndh genes, which consists of about 12 subunits and contributes to a proton gradient (DmHþ) across the membrane. The second type of dehydrogenase is a NADH-type II dehydrogenase (NDH-2) consisting of a single subunit and probably not contributing to energy transduction.

698

RESPIRATORY PROCESSES IN ANOXYGENIC AND OXYGENIC PHOTOTROPHS

NAD(P)H

O2 CN– azide

NAD(P)H deh type I and II Cyt b6/f

PQ

complex

ARTO Cyt c -553

Cyt c ox

Succinate deh

Succinate CM

ICM NAD(P)H deh Succinate

type I and II

hn

Succinate deh PC PS II

H 2O

Cyt b6/f

PQ

complex

PS I

or Cyt c -553

HQNO O2

PCP

hn

NADP COX

Cyd(QOX)

CN– azide O2

FIGURE 3 Working model of photosynthetic and respiratory electron transport chains in Synechocystis sp. strain PCC 6803 in CM and ICM. Membrane-bound redox complexes are indicated as rectangular boxes. Dotted arrows (CM redox chain) symbolize the lack of evidence for electron flow while the thickness of arrows (ICM redox chain) symbolize their relative activities. HQNO, 2-heptyl-4-hydroxy-quinoline-N-oxide; PCP, pentachlorophenol; CN2, cyanide anion; PC, plastocyanin; PQ, plastoquinone pool; hn, radiant energy; see text for further details.

In Synechocystis sp. strain PCC 6803, three genes coding for NDH-2 (ndbA,B,C) are found. Three intermingling pathways are dominant in Synechocystis sp. strain PCC 6803 thylakoids (ICM), namely: (1) a linear electron transport from H2O to NADPH, catalyzed by photosystems I and II (PSI/PSII), (2) a respiratory transport from NAD(P)H and succinate to both COX and QOX, and (3) a cyclic electron flow around PSI, i.e. electrons at the acceptor side of PSI returning to the PQ pool (Figure 3). However, as generally seen in facultative anoxygenic phototrophs, electrons can move from one pathway to another at the level of the PQ pool, b6/f complex, and/ or soluble carriers such as plastocyanin (PC) or cyt c-553 (Figure 3). For example, in the absence of PSI, reducing equivalents generated by PSII are fed into COX, while in darkness, respiratory electrons are used to reduce the acceptor side of PSII if terminal oxidases are blocked. Results with mutants of Synechocystis sp. strain PCC 683 impaired in several combinations of respiratory and photosynthetic redox complexes

indicate that succinate dehydrogenase (SDH) is the main electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases might simply operate as regulators of NADP and NAD reduction levels. This indicates that respiration in cyanobacteria plays an important role in the control of the intracellular redox balance. In general, the genes for components of the respiratory chain(s) are present in only one copy per chromosome even in those species having two respiratory chains (Figure 3). How one gene directs its gene product into two different membranes (CM and ICM) is an intriguing yet unanswered question. Further, the amounts of several components of the respiratory chain(s) are regulated by external factors such as the concentration of Cu2þ for synthesis of cyt c-553 and plastocyanin or the ionic strength for cyt aa3-type oxidase synthesis. Unfortunately, the mechanisms of gene regulation are largely unknown at present and they will be important topics for future studies in respiration of oxygenic phototrophs.

RESPIRATORY PROCESSES IN ANOXYGENIC AND OXYGENIC PHOTOTROPHS

SEE ALSO THE FOLLOWING ARTICLES Cytochrome bc 1 Complex (Respiratory Chain Complex III) † Photosynthesis † Respiratory Chain Complex II and Succinate:Quinone Oxidoreductases

GLOSSARY cytochromes Redox proteins with an iron-containing porphyrin ring (heme). electrochemical proton gradient It defines the membrane energized state in terms of electrical units. genetic regulatory element A molecule that regulates gene expression by interacting with DNA. phototroph An organism that converts radiant energy into chemical energy. proton motive force An energized state of the membrane resulting from the separation of charges across the membrane. respiration The process in which a compound is biologically oxidized by an electron acceptor (O2 or an O2 substitute) linked to generation of a proton motive force.

FURTHER READING Schmetterer, G. (1995). Cyanobacterial respiration. In The Molecular Biology of Cyanobacteria (D. A. Bryant, ed.) Vol. 1, pp. 409–435. Kluwer, Dordrecht.

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Vermeglio, A., Borghese, R., and Zannoni, D. (2004). Interaction between photosynthesis and respiration in facultative phototrophs. In Respiration in Archaea and Bacteria (D. Zannoni, ed.) Kluwer, Dordrecht, Vol. 16. Zannoni, D. (1995). Aerobic and anaerobic electron transport chains in anoxygenic phototrophic bacteria. In Anoxygenic Photosynthetic Bacteria (R. E. Blankenship, M. T. Madigan and C. E. Bauer, eds.) Vol. 2, pp. 949–971. Kluwer, Dordrecht.

BIOGRAPHY Davide Zannoni is Professor of General Microbiology in the Department of Biology at the University of Bologna, Italy. His main research interests are in the broad field of microbial physiology and biochemistry of facultative phototrophic and aerobic bacteria. He holds a degree in biology from the University of Bologna and received postdoctoral training at the St. Louis University School of Medicine. He carried out pioneering studies on bioenergetics of bacterial respiration and interaction between photosynthetic and respiratory redox complexes in facultative phototrophs. Roberto Borghese is Research Associate and Lecturer of General Microbiology in the Department of Biology at the University of Bologna, Italy. He holds a degree in biology from the University of Bologna and a Ph.D. from the University of Missouri – Columbia. His main research interest is in genetics of anoxygenic photosynthetic bacteria.