Rieske Iron-sulfur Protein Research Articles

Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6f complex.

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.

The nematode effector Mj-NEROSs interacts with Rieske's iron-sulfur protein influencing plastid ROS production to suppress plant immunity.

Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process.

Rieske Iron-Sulfur Protein Mediates Pulmonary Hypertension Following Nicotine/Hypoxia Coexposure.

The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle-specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated-mediated DNA damage and NF-κB-dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress-associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications.

The phylogeny of Acetobacteraceae : photosynthetic traits and deranged respiratory enzymes

ABSTRACT We present here a comprehensive phylogenomic analysis of Acetobacteraceae , a vast group of alphaproteobacteria that has been widely studied for their economic importance. Our results indicate that the ancestor of Acetobacteraceae most likely was photosynthetic and evolved via a progressive transition from versatile photoferrotrophy to the incomplete oxidation of organic substrates defining acetous physiology. Vestigial signs of photosynthetic carotenoid metabolism are present in non-photosynthetic acetous taxa that have lost cytochrome oxidase, while their sister taxa retain such traits. The dominant terminal oxidase of acetous bacteria, the bo 3 ubiquinol oxidase, is derived from duplication and diversification of operons present in Acidocella taxa that have lost photosynthesis. We analyzed the bioenergetic traits that can compensate for the electron transfer function of photosynthetic reaction centers or constitute alternative pathways for the oxidoreduction of c -type cytochromes, such as iron oxidation. The latter pathway bypasses the deranged cytochrome bc 1 complex that is characteristically present in acidophilic taxa due to the loss of conserved ligands in both the Rieske iron-sulfur protein and cytochrome b subunit. The deranged or non-functional bc 1 complex may be retained for its structural role in stabilizing Complex I. The combination of our phylogenetic analysis with in-depth functional evaluations indicates that the order Acetobacterales needs to be emended to include three families: Acetobacteraceae sensu stricto , Roseomonadaceae fam. nov., and Acidocellaceae fam. nov. IMPORTANCE Acetobacteraceae are one of the best known and most extensively studied groups of bacteria, which nowadays encompasses a variety of taxa that are very different from the vinegar-producing species defining the family. Our paper presents the most detailed phylogeny of all current taxa classified as Acetobacteraceae , for which we propose a taxonomic revision. Several of such taxa inhabit some of the most extreme environments on the planet, from the deserts of Antarctica to the Sinai desert, as well as acidic niches in volcanic sites like the one we have been studying in Patagonia. Our work documents the progressive variation of the respiratory chain in early branching Acetobacteraceae into the different respiratory chains of acidophilic taxa such as Acidocella and acetous taxa such as Acetobacter . Remarkably, several genomes retain remnants of ancestral photosynthetic traits and functional bc 1 complexes. Thus, we propose that the common ancestor of Acetobacteraceae was photosynthetic.

A concerted ATPase cycle of the protein transporter AAA-ATPase Bcs1

Bcs1, a homo-heptameric transmembrane AAA-ATPase, facilitates folded Rieske iron-sulfur protein translocation across the inner mitochondrial membrane. Structures in different nucleotide states (ATPγS, ADP, apo) provided conformational snapshots, but the kinetics and structural transitions of the ATPase cycle remain elusive. Here, using high-speed atomic force microscopy (HS-AFM) and line scanning (HS-AFM-LS), we characterized single-molecule Bcs1 ATPase cycling. While the ATP conformation had ~5600 ms lifetime, independent of the ATP-concentration, the ADP/apo conformation lifetime was ATP-concentration dependent and reached ~320 ms at saturating ATP-concentration, giving a maximum turnover rate of 0.17 s−1. Importantly, Bcs1 ATPase cycle conformational changes occurred in concert. Furthermore, we propose that the transport mechanism involves opening the IMS gate through energetically costly straightening of the transmembrane helices, potentially driving rapid gate resealing. Overall, our results establish a concerted ATPase cycle mechanism in Bcs1, distinct from other AAA-ATPases that use a hand-over-hand mechanism.

The cytochrome b6f complex: plastoquinol oxidation and regulation of electron transport in chloroplasts.

In oxygenic photosynthetic systems, the cytochrome b6f (Cytb6f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb6f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH2). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH2 from PSII to the Cytb6f does not limit the intersystem electron transport. The overall rate of PQH2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH2 at the catalytic site Qo, i.e., the reaction of electron transfer from PQH2 to the Fe2S2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH2 oxidation within the framework of a model system including the Fe2S2 cluster of the ISP and surrounding amino acids, the low-potential heme b6L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH2). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb6f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

Mitochondrial Rieske iron-sulfur protein is a crucial molecule in COPD-associated pulmonary hypertension

Pulmonary hypertension (PH) often occurs in chronic obstructive pulmonary disease (COPD) and is the major cause of death in COPD patients. Molecular mechanisms for PH in COPD remain poorly understood, and clinical treatments are neither specific nor effective. Cigarette and e-cigarette smoking (CS) account for up to 90% of COPD cases, in which nicotine is the major active component and causes COPD-like cellular responses; moreover, hypoxia is a known key factor in the development of PH. In this study, we aim to develop a PH in COPD animal model and determine the underlying molecular mechanisms. Our findings revealed that co-exposure of nicotine (or CS) and hypoxia synergistically elevated PH (i.e., right ventricular pressure and weight) compared to nicotine, CS, or hypoxia alone. We also found that mitochondrial reactive oxygen species ([ROS] m ), assessed using mitochondrial-specific ROS indicator MitoSOX, was significantly increased in pulmonary artery smooth muscle cells (PASMCs) from CS patients with COPD compared to normal PASMCs from non-CS patients. The increased [ROS] m was blocked by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Rieske iron-sulfur protein (RISP) in mitochondrial complex III. More importantly, SMC-specific RISP gene knockout blocked nicotine/hypoxia exposure-induced PA remodeling and hypertension, whereas SMC-specific RISP overexpression exacerbated PH in mice. We further discovered that RISP regulated DNA damage following nicotine/hypoxia co-exposure via ataxia telangiectasia-mutated (ATM) signaling. RISP also mediated nuclear factor (NF)-κB inflammatory signaling. Together, our studies for the first time demonstrate that nicotine/hypoxia co-exposure leads to RISP-dependent [ROS] m , ATM-mediated DNA damage-dependent NF-κB-associated inflammation, PA vasoremodeling and PH in COPD in a sex-dependent manner. Moreover, RISP, ATM, and NF-κB may serve as novel and effective molecular targets in treating COPD and its associated PH. NIH R01 HL122865, NIH R03AG070784, and NIH R01 HL108232 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The ATPase cycle of the AAA-ATPase protein transporter Bcs1 is highly concerted.

Bcs1, a homo-heptameric transmembrane AAA-ATPase that belongs to the superfamily of AAA proteins, acts as translocation machinery for the folded Rieske iron-sulfur protein (ISP, a key subunit of the mitochondrial complex-III) across the inner mitochondrial membrane. Cryo-EM structures depicted a ring-shaped transporter with two cavities, a cylindrical one formed by the ATPase domains on the matrix surface and a conical one formed by the transmembrane helices. Structures in different nucleotide states (ATPγS, ADP, APO) provided intuitive snapshots of the functional cycle, but the kinetics and structural transitions of the ATPase cycle remain entirely unknown. Here, using high-speed atomic force microscopy (HS-AFM) and HS-AFM line scanning (HS-AFM-LS), we characterized the ATPase cycle of Bcs1 at the single molecule level with sub-millisecond temporal resolution. We determined, in conditions where the molecules are in a continuous ATPase cycle turnover, that the ATP-bound conformational has a lifetime of ∼3700 ms while the lifetime of ADP/APO conformation is much shorter, ∼400 ms. By monitoring the conformational state of the two Bcs1 protomers on the two opposite sides of the ring at microsecond temporal resolution, we found that ATP-binding and -hydrolysis coupled conformational changes are highly concerted. We propose that the opening of the cone cavity to the intermembrane surface through the straightening of the transmembrane helices in the membrane is energetically costly and membrane elasticity drives the resealing of the gate. Overall, our results establish a working mechanism for the ATPase cycle of Bcs1 at the single molecule level and propose a mechanism for how Bcs1 can translocate a folded protein.

Role of ryanodine receptor 2 and FK506-binding protein 12.6 dissociation in pulmonary hypertension.

Pulmonary hypertension (PH) is a devastating disease characterized by a progressive increase in pulmonary arterial pressure leading to right ventricular failure and death. A major cellular response in this disease is the contraction of smooth muscle cells (SMCs) of the pulmonary vasculature. Cell contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i), which is generated and regulated by various ion channels. Several studies by us and others have shown that ryanodine receptor 2 (RyR2), a Ca2+-releasing channel in the sarcoplasmic reticulum (SR), is an essential ion channel for the control of [Ca2+]i in pulmonary artery SMCs (PASMCs), thereby mediating the sustained vasoconstriction seen in PH. FK506-binding protein 12.6 (FKBP12.6) strongly associates with RyR2 to stabilize its functional activity. FKBP12.6 can be dissociated from RyR2 by a hypoxic stimulus to increase channel function and Ca2+ release, leading to pulmonary vasoconstriction and PH. More specifically, dissociation of the RyR2-FKBP12.6 complex is a consequence of increased mitochondrial ROS generation mediated by the Rieske iron-sulfur protein (RISP) at the mitochondrial complex III after hypoxia. Overall, RyR2/FKBP12.6 dissociation and the corresponding signaling pathway may be an important factor in the development of PH. Novel drugs and biologics targeting RyR2, FKBP12.6, and related molecules may become unique effective therapeutics for PH.

Soluble domains of cytochrome c-556 and Rieske iron-sulfur protein from Chlorobaculum tepidum: Crystal structures and interaction analysis.

In photosynthetic green sulfur bacteria, the electron transfer reaction from menaquinol:cytochrome c oxidoreductase to the P840 reaction center (RC) complex occurs directly without any involvement of soluble electron carrier protein(s). X-ray crystallography has determined the three-dimensional structures of the soluble domains of the CT0073 gene product and Rieske iron-sulfur protein (ISP). The former is a mono-heme cytochrome c with an α-absorption peak at 556nm. The overall fold of the soluble domain of cytochrome c-556 (designated as cyt c-556sol) consists of four α-helices and is very similar to that of water-soluble cyt c-554 that independently functions as an electron donor to the P840 RC complex. However, the latter's remarkably long and flexible loop between the α3 and α4 helices seems to make it impossible to be a substitute for the former. The structure of the soluble domain of the Rieske ISP (Rieskesol protein) shows a typical β-sheets-dominated fold with a small cluster-binding and a large subdomain. The architecture of the Rieskesol protein is bilobal and belongs to those of b6f-type Rieske ISPs. Nuclear magnetic resonance (NMR) measurements revealed weak non-polar but specific interaction sites on Rieskesol protein when mixed with cyt c-556sol. Therefore, menaquinol:cytochrome c oxidoreductase in green sulfur bacteria features a Rieske/cytb complex tightly associated with membrane-anchored cyt c-556.

Structural heterogeneity of the Rieske iron-sulfur protein in a yeast complex III2.

Structural heterogeneity of the Rieske iron-sulfur protein in a yeast complex III2.

Analysis of the conformational heterogeneity of the Rieske iron-sulfur protein in complex III2 by cryo-EM.

Movement of the Rieske domain of the iron-sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.

Algal PETC-Pro171-Leu suppresses electron transfer in cytochrome b6f under acidic lumenal conditions.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

The Potential Important Role of Mitochondrial Rieske Iron-Sulfur Protein as a Novel Therapeutic Target for Pulmonary Hypertension in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, which is often due to pulmonary hypertension (PH). The underlying molecular mechanisms are poorly understood, and current medications are neither specific nor always effective. In this review, we highlight the recent findings on the roles of altered mitochondrial bioenergetics in PH in COPD. We also discuss the central role of mitochondrial reactive oxygen species (ROS) generation mediated by Rieske iron–sulfur protein (RISP) and review the contributions of RISP-dependent DNA damage and NF-κB-associated inflammatory signaling. Finally, the potential importance of mitochondrial RISP and its associated molecules as novel therapeutic targets for PH in COPD are meticulously discussed.

Non-rolling flag leaves use an effective mechanism to reduce water loss and light-induced damage under drought stress.

The study reports on four different types of flag leaf rolling under soil drought in relation to the level of cell wall-bound phenolics. The flag leaf colonization by aphids, as a possible bioindicator of the accumulation of cell wall-bound phenolics, was also estimated. The proteins of the photosynthetic apparatus that form its core and are crucial for maintaining its stability (D1/PsbA protein), limit destructive effects of light (PsbS, a protein binding carotenoids in the antennas) and participate in efficient electron transport between photosystems II (PSII) and PSI (Rieske iron-sulfur protein of the cytochrome b6f complex) were evaluated in two types of flag leaf rolling. Additionally, biochemical and physiological reactions to drought stress in rolling and non-rolling flag leaves were compared. The study identified four types of genome-related types of flag leaf rolling. The biochemical basis for these differences was a different number of phenolic molecules incorporated into polycarbohydrate structures of the cell wall. In an extreme case of non-rolling dehydrated flag leaves, they were found to accumulate high amounts of cell wall-bound phenolics that limited cell water loss and protected the photosynthetic apparatus against excessive light. PSII was also additionally protected against excess light by the accumulation of photosynthetic apparatus proteins that ensured stable and efficient transport of excitation energy beyond PSII and its dissipation as far-red fluorescence and heat. Our analysis revealed a new type of flag leaf rolling brought about by an interaction between wheat and rye genomes, and resulting in biochemical specialization of flexible, rolling and rigid, non-rolling parts of the flag leaf. The study confirmed limited aphid colonization of the flag leaves with enhanced content of cell wall-bound phenolics. Non-rolling leaves developed effective adaptation mechanisms to reduce both water loss and photoinhibitory damage to the photosynthetic apparatus under drought stress.

Important Functions and Molecular Mechanisms of Mitochondrial Redox Signaling in Pulmonary Hypertension

Mitochondria are important organelles that act as a primary site to produce reactive oxygen species (ROS). Additionally, mitochondria play a pivotal role in the regulation of Ca2+ signaling, fatty acid oxidation, and ketone synthesis. Dysfunction of these signaling molecules leads to the development of pulmonary hypertension (PH), atherosclerosis, and other vascular diseases. Features of PH include vasoconstriction and pulmonary artery (PA) remodeling, which can result from abnormal proliferation, apoptosis, and migration of PA smooth muscle cells (PASMCs). These responses are mediated by increased Rieske iron–sulfur protein (RISP)-dependent mitochondrial ROS production and increased mitochondrial Ca2+ levels. Mitochondrial ROS and Ca2+ can both synergistically activate nuclear factor κB (NF-κB) to trigger inflammatory responses leading to PH, right ventricular failure, and death. Evidence suggests that increased mitochondrial ROS and Ca2+ signaling leads to abnormal synthesis of ketones, which play a critical role in the development of PH. In this review, we discuss some of the recent findings on the important interactive role and molecular mechanisms of mitochondrial ROS and Ca2+ in the development and progression of PH. We also address the contributions of NF-κB-dependent inflammatory responses and ketone-mediated oxidative stress due to abnormal regulation of mitochondrial ROS and Ca2+ signaling in PH.

ATPase cycle of Bcs1 AAA-ATPase characterized by high-speed AFM at millisecond time resolution

Bcs1 belongs to the superfamily of AAA proteins, ring-shaped homo-oligomeric ATPases associated with numerous cellular activities including protein unfolding and degradation, DNA replication and membrane fusion. Different from many AAA proteins, Bcs1 is a transmembrane protein, a homo-heptamer, that has been demonstrated to act as translocation machinery for the folded Rieske iron-sulfur protein (ISP, a key subunit of the mitochondrial complex-III) across the inner mitochondrial membrane. In general, canonical AAA proteins utilize a hand-over-hand mechanism, where the individual protomer interchange the three different nucleotide states (ATP, ADP, APO) in a sequential manner.

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C.glutamicum supercomplex and used cryo-EM to determine its structure at 2.9Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of "innate-like" T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1-/- ), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1-/- mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1-/- mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.