Mutations in the UQCC1-Interacting Protein,UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.     

Biogenesis of the bc1 Complex of the Mitochondrial Respiratory Chain

The oxidative phosphorylation system contains four respiratory chain complexes that connect the transport of electrons to oxygen with the establishment of an electrochemical gradient over the inner membrane for ATP synthesis. Due to the dual genetic source of the respiratory chain subunits, its assembly requires a tight coordination between nuclear and mitochondrial gene expression machineries. In addition, dedicated assembly factors support the step-by-step addition of catalytic and accessory subunits as well as the acquisition of redox cofactors. Studies in yeast have revealed the basic principles underlying the assembly pathways. In this review, we summarize work on the biogenesis of the bc₁ complex or complex III, a central component of the mitochondrial energy conversion system.     

Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy

Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b₅ (cytb₅) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb₅ interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb₅ at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb₅ resonances characterize CYP2B4-cytb₅ interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb₅, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb₅ imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb₅ complex formation by orienting tr-CYP2B4 for efficient contact with cytb₅. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb₅ in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb₅ complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.     

Assembly of a transmembrane b-Type cytochrome is mainly driven by transmembrane helix interactions

Folding, assembly and stability of α-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b₅₅₉^′ was studied by fluorescence resonance energy transfer. Cytochrome b₅₅₉^′ consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b₅₅₉^′ monomer, each carrying a specific fluorescent dye, allowed monitoring helix-helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.     

Superoxide production by cytochrome bc1 complex: A mathematical model

Reactive oxygen species (ROS) are involved in the pathophysiology of several diseases (e.g. Alzheimer or atherosclerosis) and also in the aging process. The main source of ROS in aerobic organisms is the electron transport chain (ETC) in the inner mitochondrial membrane. Superoxide is produced at complexes I and III of the ETC, starting a complex network of ROS reactions. To achieve a deeper mechanistic understanding of how ROS are generated by complex III, we developed a mathematical model that successfully describes experimental data of complex III activity in various rat tissues, the production of ROS with and without antimycin and ROS generation depending on different values of the membrane potential ?Ψ. The model also reinforces the idea of ubiquinone acting as a redox mediator between heme b_L and oxygen, as proposed earlier.     

Role of the Twin Arginine Protein Transport Pathway in the Assembly of the Streptomyces coelicolor Cytochrome bc1 Complex

The cytochrome bc₁-cytochrome aa₃ complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc₁ complex shows a number of unusual features in comparison to other cytochrome bc₁ complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc₁ complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ΔtatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc₁ complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc₁ complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.     

Molecular mechanism of metabolic NAD(P)H-dependent electron-transfer systems: The role of redox cofactors

NAD(P)H-dependent electron-transfer (ET) systems require three functional components: a flavin-containing NAD(P)H-dehydrogenase, one-electron carrier and metal-containing redox center. In principle, these ET systems consist of one-, two- and three-components, and the electron flux from pyridine nucleotide cofactors, NADPH or NADH to final electron acceptor follows a linear pathway: NAD(P)H?→?flavin?→?one-electron carrier?→?metal containing redox center. In each step ET is primarily controlled by one- and two-electron midpoint reduction potentials of protein-bound redox cofactors in which the redox-linked conformational changes during the catalytic cycle are required for the domain-domain interactions. These interactions play an effective ET reactions in the multi-component ET systems. The microsomal and mitochondrial cytochrome P450 (cyt P450) ET systems, nitric oxide synthase (NOS) isozymes, cytochrome b₅ (cyt b₅) ET systems and methionine synthase (MS) ET system include a combination of multi-domain, and their organizations display similarities as well as differences in their components. However, these ET systems are sharing of a similar mechanism. More recent structural information obtained by X-ray and cryo-electron microscopy (cryo-EM) analysis provides more detail for the mechanisms associated with multi-domain ET systems. Therefore, this review summarizes the roles of redox cofactors in the metabolic ET systems on the basis of one-electron redox potentials. In final Section, evolutionary aspects of NAD(P)H-dependent multi-domain ET systems will be discussed.     

Biogenesis of respiratory cytochromes in bacteria.

Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.     

The tRNA Splicing Endonuclease Complex Cleaves the Mitochondria-localized CBP1 mRNA

The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652–726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.     

Ubiquinol oxidation in the cytochrome bc1 complex: Reaction mechanism and prevention of short-circuiting

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc₁ complex (bc₁). This integral membrane complex serves as a “hub” in the vast majority of electron transfer chains. The bc₁ oxidizes a ubiquinol molecule to ubiquinone by a unique “bifurcated” reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe-2S] iron-sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc₁, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc₁ and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc₁ is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria

Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (M_r 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (E_m = ?224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Studies on the cytochrome b6/f complex. I. Characterization of the complex subunits in Chlamydomonas reinhardtii

We have analyzed the heme-associated peroxidase activity in thylakoid membranes from the green algae Chlamydomonas reinhardtii after electrophoresis in the presence of sodium dodecyl sulfate. Besides cytochrome f and cytochrome b₆, we observed peroxidase activity in two other bands, of 34 and 11 kDa, of unknown origin. Characterization of the b₆/f complex subunits was undertaken by means of a comparison of the polypeptide deficiencies in several b₆/f mutants with the polypeptide content of preparations enriched in b₆/f complexes. We conclude that the b₆/f complex consists of five subunits. Using site-specific translation inhibitors, we show that cytochrome f, cytochrome b₆ and subunit IV are of chloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmic ribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunits of chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of the subunits encoded in the nuclear genome.     

Quinone Pathways in Entire Photosynthetic Chromatophores of Rhodospirillum photometricum

In photosynthetic organisms, membrane pigment-protein complexes [light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2)] harvest solar energy and convert sunlight into an electrical and redox potential gradient (reaction center) with high efficiency. Recent atomic force microscopy studies have described their organization in native membranes. However, the cytochrome (cyt) bc₁ complex remains unseen, and the important question of how reduction energy can efficiently pass from core complexes (reaction center and LH1) to distant cyt bc₁ via membrane-soluble quinones needs to be addressed. Here, we report atomic force microscopy images of entire chromatophores of Rhodospirillum photometricum. We found that core complexes influence their molecular environment within a critical radius of ∼ 250 Å. Due to the size mismatch with LH2, lipid membrane spaces favorable for quinone diffusion are found within this critical radius around cores. We show that core complexes form a network throughout entire chromatophores, providing potential quinone diffusion pathways that will considerably speed the redox energy transfer to distant cyt bc₁. These long-range quinone pathway networks result from cooperative short-range interactions of cores with their immediate environment.     

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus

The cyanobacterial cytochrome b₆f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b₆f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700⁺ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b₆f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b₆f complexes with different functions that might be correlated with supercomplex formation.