COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

An organic O donor for biological hydroxylation reactions.

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2, or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.

Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2.

Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.

Diversification of Ubiquinone Biosynthesis via Gene Duplications, Transfers, Losses, and Parallel Evolution.

The availability of an ever-increasing diversity of prokaryotic genomes and metagenomes represents a major opportunity to understand and decipher the mechanisms behind the functional diversification of microbial biosynthetic pathways. However, it remains unclear to what extent a pathway producing a specific molecule from a specific precursor can diversify. In this study, we focus on the biosynthesis of ubiquinone (UQ), a crucial coenzyme that is central to the bioenergetics and to the functioning of a wide variety of enzymes in Eukarya and Pseudomonadota (a subgroup of the formerly named Proteobacteria). UQ biosynthesis involves three hydroxylation reactions on contiguous carbon atoms. We and others have previously shown that these reactions are catalyzed by different sets of UQ-hydroxylases that belong either to the iron-dependent Coq7 family or to the more widespread flavin monooxygenase (FMO) family. Here, we combine an experimental approach with comparative genomics and phylogenetics to reveal how UQ-hydroxylases evolved different selectivities within the constrained framework of the UQ pathway. It is shown that the UQ-FMOs diversified via at least three duplication events associated with two cases of neofunctionalization and one case of subfunctionalization, leading to six subfamilies with distinct hydroxylation selectivity. We also demonstrate multiple transfers of the UbiM enzyme and the convergent evolution of UQ-FMOs toward the same function, which resulted in two independent losses of the Coq7 ancestral enzyme. Diversification of this crucial biosynthetic pathway has therefore occurred via a combination of parallel evolution, gene duplications, transfers, and losses.

Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis, by Adrenodoxin Reductase/Ferredoxin.

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in-depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by decreasing NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis-Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half-reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH-AdxR-Yah1p-Coq6p, where Yah1p shuttles electrons between AdxR and Coq6p. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.

Structural Reconstruction of E. coli Ubi Metabolon Using an AlphaFold2-Based Computational Framework.

Ubiquinone (UQ) is a redox polyisoprenoid lipid found in the membranes of bacteria and eukaryotes that has important roles, notably one in respiratory metabolism, which sustains cellular bioenergetics. In Escherichia coli, several steps of the UQ biosynthesis take place in the cytosol. To perform these reactions, a supramolecular assembly called Ubi metabolon is involved. This latter is composed of seven proteins (UbiE, UbiG, UbiF, UbiH, UbiI, UbiJ, and UbiK), and its structural organization is unknown as well as its protein stoichiometry. In this study, a computational framework has been designed to predict the structure of this macromolecular assembly. In several successive steps, we explored the possible protein interactions as well as the protein stoichiometry, to finally obtain a structural organization of the complex. The use of AlphaFold2-based methods combined with evolutionary information enabled us to predict several models whose quality and confidence were further analyzed using different metrics and scores. Our work led to the identification of a "core assembly" that will guide functional and structural characterization of the Ubi metabolon.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

Towards Molecular Understanding of the Functional Role of UbiJ-UbiK2 Complex in Ubiquinone Biosynthesis by Multiscale Molecular Modelling Studies.

Ubiquinone (UQ) is a polyisoprenoid lipid found in the membranes of bacteria and eukaryotes. UQ has important roles, notably in respiratory metabolisms which sustain cellular bioenergetics. Most steps of UQ biosynthesis take place in the cytosol of E. coli within a multiprotein complex called the Ubi metabolon, that contains five enzymes and two accessory proteins, UbiJ and UbiK. The SCP2 domain of UbiJ was proposed to bind the hydrophobic polyisoprenoid tail of UQ biosynthetic intermediates in the Ubi metabolon. How the newly synthesised UQ might be released in the membrane is currently unknown. In this paper, we focused on better understanding the role of the UbiJ-UbiK2 heterotrimer forming part of the metabolon. Given the difficulties to gain functional insights using biophysical techniques, we applied a multiscale molecular modelling approach to study the UbiJ-UbiK2 heterotrimer. Our data show that UbiJ-UbiK2 interacts closely with the membrane and suggests possible pathways to enable the release of UQ into the membrane. This study highlights the UbiJ-UbiK2 complex as the likely interface between the membrane and the enzymes of the Ubi metabolon and supports that the heterotrimer is key to the biosynthesis of UQ8 and its release into the membrane of E. coli.

Recent advances in the metabolic pathways and microbial production of coenzyme Q.

Coenzyme Q (CoQ) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. Here, we review discovery of the pathway with a particular focus on its superstructuration and regulation, and we summarize the metabolic engineering strategies for overproduction of CoQ by microorganisms. Studies in model microorganisms elucidated the details of CoQ biosynthesis and revealed the existence of multiprotein complexes composed of several enzymes that catalyze consecutive reactions in the CoQ pathways of Saccharomyces cerevisiae and Escherichia coli. Recent findings indicate that the identity and the total number of proteins involved in CoQ biosynthesis vary between species, which raises interesting questions about the evolution of the pathway and could provide opportunities for easier engineering of CoQ production. For the biotechnological production, so far only microorganisms have been used that naturally synthesize CoQ10 or a related CoQ species. CoQ biosynthesis requires the aromatic precursor 4-hydroxybenzoic acid and the prenyl side chain that defines the CoQ species. Up to now, metabolic engineering strategies concentrated on the overproduction of the prenyl side chain as well as fine-tuning the expression of ubi genes from the ubiquinone modification pathway, resulting in high CoQ yields. With expanding knowledge about CoQ biosynthesis and exploration of new strategies for strain engineering, microbial CoQ production is expected to improve.

The Biosynthetic Pathway of Ubiquinone Contributes to Pathogenicity of Francisella novicida.

Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp. are strict aerobes, and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli, allowing the identification of 15 Ubi proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified ubiquinone 8 (UQ8) as the major quinone found in the membranes of this bacterium. Next, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics, and biochemical approaches. Our analysis disclosed the presence in Francisella of 10 putative Ubi proteins, and we confirmed 8 of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share similar patterns. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp., and homologs of the Ubi proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Next, via two approaches, i.e., the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, both of which strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. IMPORTANCE Francisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We show that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor that strongly decreased the biosynthesis of UQ. Our demonstration of the crucial roles of UQ for the respiratory metabolism of F. novicida and for the involvement in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.

Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency.

COQ2 (p-hydroxybenzoate polyprenyl transferase) encodes the enzyme required for the second step of the final reaction sequence of Coenzyme Q10 (CoQ) biosynthesis. Its mutations represent a frequent cause of primary CoQ deficiency and have been associated with the widest clinical spectrum, ranging from fatal neonatal multisystemic disease to late-onset encephalopathy. However, the reasons of this variability are still unknown.We have characterized the structure of human COQ2, defined its subcellular localization and developed a yeast model to validate all the mutant alleles reported so far.Our findings show that the main functional transcript of COQ2 is shorter than what was previously reported and that its protein product localizes to mitochondria with the C-terminus facing the intermembrane space. Complementation experiments in yeast showed that the residual activity of the mutant proteins correlates with the clinical phenotypes observed in patients.We defined the structure of COQ2 with relevant implications for mutation screening in patients and demonstrated that, contrary to other COQ gene defects such as ADCK3, there is a correlation between COQ2 genotype and patient's phenotype.

Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6.

Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

Coq6 is responsible for the C4-deamination reaction in coenzyme Q biosynthesis in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid as a precursor of coenzyme Q, a redox lipid essential to the function of the mitochondrial respiratory chain. The biosynthesis of coenzyme Q from pABA requires a deamination reaction at position C4 of the benzene ring to substitute the amino group with an hydroxyl group. We show here that the FAD-dependent monooxygenase Coq6, which is known to hydroxylate position C5, also deaminates position C4 in a reaction implicating molecular oxygen, as demonstrated with labeling experiments. We identify mutations in Coq6 that abrogate the C4-deamination activity, whereas preserving the C5-hydroxylation activity. Several results support that the deletion of Coq9 impacts Coq6, thus explaining the C4-deamination defect observed in Δcoq9 cells. The vast majority of flavin monooxygenases catalyze hydroxylation reactions on a single position of their substrate. Coq6 is thus a rare example of a flavin monooxygenase that is able to act on two different carbon atoms of its C4-aminated substrate, allowing its deamination and ultimately its conversion into coenzyme Q by the other proteins constituting the coenzyme Q biosynthetic pathway.

Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site.

Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.

Biosynthesis and physiology of coenzyme Q in bacteria.

Ubiquinone, also called coenzyme Q, is a lipid subject to oxido-reduction cycles. It functions in the respiratory electron transport chain and plays a pivotal role in energy generating processes. In this review, we focus on the biosynthetic pathway and physiological role of ubiquinone in bacteria. We present the studies which, within a period of five decades, led to the identification and characterization of the genes named ubi and involved in ubiquinone production in Escherichia coli. When available, the structures of the corresponding enzymes are shown and their biological function is detailed. The phenotypes observed in mutants deficient in ubiquinone biosynthesis are presented, either in model bacteria or in pathogens. A particular attention is given to the role of ubiquinone in respiration, modulation of two-component activity and bacterial virulence. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency.

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast Δcoq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients.

Three conserved histidine residues contribute to mitochondrial iron transport through mitoferrins.

Iron is an essential element for almost all organisms. In eukaryotes, it is mainly used in mitochondria for the biosynthesis of iron-sulfur clusters and haem group maturation. Iron is delivered into the mitochondrion by mitoferrins, members of the MCF (mitochondrial carrier family), through an unknown mechanism. In the present study, the yeast homologues of these proteins, Mrs3p (mitochondrial RNA splicing 3) and Mrs4p, were studied by inserting them into liposomes. In this context, they could transport Fe2+ across the proteoliposome membrane, as shown using the iron chelator bathophenanthroline. A series of amino acid-modifying reagents were screened for their effects on Mrs3p-mediated iron transport. The results of the present study suggest that carboxy and imidazole groups are essential for iron transport. This was confirmed by in vivo complementation assays, which demonstrated that three highly conserved histidine residues are important for Mrs3p function. These histidine residues are not conserved in other MCF members and thus they are likely to play a specific role in iron transport. A model describing how these residues help iron to transit smoothly across the carrier cavity is proposed and compared with the structural and biochemical data available for other carriers in this family.