Manganese Cofactors Research Articles

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography.

Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.

Utilization of the acid hydrolysate of defatted Chlorella biomass as a sole fermentation substrate for the production of biosurfactant from Bacillus subtilis C9

Although algal biofuels have been promoted as a carbon-neutral alternative to fossil fuels, several economic and technological barriers challenge the much-anticipated success of algae as a commercial bio-feedstock. In particular, utilizing only a lipid portion of harvested algal biomass limits possible monetary returns from algal biomass within which non-lipid compounds generally comprise a major proportion. Previous studies thus demonstrated the production of ethanol from defatted algal biomass to improve overall economic outlook of the nascent algae industry; however, it is also possible to utilize residual algal biomass as a fermentation substrate for bacterial strains producing high value alternative products. Therefore, the goal of this study was to compare the yield of biosurfactant from B. subtilis C9 in conventional culture medium and the acid hydrolysate of defatted Chlorella biomass (DCB). Although increasing the concentration of acid catalyst from 0 N to 1.0 N was expected to proportionally enhance the effectiveness of dilute acid hydrolysis, the results indicated that DCB hydrolysates treated under different acid conditions at 121 °C for 15 min exhibited the highest yield of fermentable monosaccharides when treated with 0.25 N HCl. Subsequently, the fermentation of B. subtilis C9 was performed in both Luria-Bertani (LB) and DCB hydrolysate medium after adjusting salinity and monosaccharide concentration to identical levels; a significantly greater yield of biosurfactant was observed in the culture grown with DCB hydrolysate medium, while the presence of magnesium and manganese co-factors in DCB hydrolysate contributed to a greater biosurfactant yield. Collectively, these results suggest that the valorization of defatted algal biomass could be also accomplished by incorporating biosurfactant fermentation into algal biorefinery processes and call for further process optimization efforts that consider factors associated with the operation of industrial algal biorefineries.

Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

CH‐π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

AbstractThe class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4‐dihydroxy‐α‐ketoacids. The crystal structure of holo‐SwHKA in complex with hydroxypyruvate revealed CH‐π interactions between the C−H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent C−C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β)8 barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA.magnified image

Merged Heme and Non-Heme Manganese Cofactors for a Dual Antioxidant Surveillance in Photosynthetic Organisms

The coupling of a polycationic Mn(III)-porphyrin, with a dinuclear Mn2(II,II)L2 core (HL = 2-{[[di(2-pyridyl)methyl](methyl)amino]methyl}phenol), results in a dual Superoxide Dismutase (SOD) and Catalase (CAT) functional analogue, Mn2L2Pn+, enabling a detoxification cascade of the superoxide anion and hydrogen peroxide into benign H2O and O2. The SOD/CAT artificial manifolds, joined in one asset, exhibit a peak catalytic performance under physiological conditions, with log kcat(O2• –) ≥ 7 and kcat(H2O2)/KM = 1890. The dual-enzyme (dizyme) concept allows for a built-in self-protection against the irreversible bleaching of the porphyrin unit (>75% protection), readily induced by H2O2 (200 μM, 20 equiv, in buffer solution, pH 7.8). We show herein that incubation of the photosynthetic green algae, Chlamydomonas reinhardtii, with the synthetic dizyme (as low as 0.1 μM), prevents H2O2 accumulation under high-light illumination conditions, thus providing antioxidant surveillance and photoprotection.

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study.

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(ii) and manganese(ii) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe(II) as a Lewis acid under normal growth conditions but which switch to Mn(II) under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe(II) and Mn(II), the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, "discrimination" between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.

Cellular Iron Distribution in Bacillus anthracis

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.

Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo

Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.

Energetics for Oxidation of a Bound Manganese Cofactor in Modified Bacterial Reaction Centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn2+ with a dissociation constant of 1 μM at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P+ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn2+ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P+. The extent of P+ reduction was found to systematically range from a full reduction in the mutants with high P/P+ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn2+ oxidation on the P/P+ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn2+/Mn3+ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn2+/Mn3+ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn2+ oxidation ranging from 30 to 120 s(-1) as the P/P+ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s(-1), reflecting an increase in the free energy difference due to the lower Mn2+/Mn3+ midpoint potential. This dependence of the rate constant on the P/P+ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s(-1) and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn4Ca cofactor of photosystem II.

Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2'O-methyltransferase that marks the 3' terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3' dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3' terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 3' nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.

Functional chemical models of multielectron water oxidation in photosynthesis

The study of water oxidation by Mn n IV clusters, which are functional chemical models of the manganese cofactor (enzyme that oxidizes water in photosystem II of natural photosynthesis) has demonstrated that a Mn 2 IV cluster oxidizes two water molecules to form one oxygen molecule, Mn 4 IV oxidizes four water molecules to form two O2 molecules, and Mn 8 IV oxidizes eight water molecules to form four O2 molecules. A Mn 6 IV cluster oxidizes six water molecules to two ozone molecules, whereas Mn 12 IV oxidizes twelve water molecules to four ozone molecules. The six-electron oxidation of water to ozone is also observed in photosynthesis in red and brown sea algae under conditions of water deficit. It is hypothesized that, in the case of water deficit in algae, the manganese cofactor with four manganese ions turns into the cofactor with six manganese ions.

Photogeneration of ozone by marine algae and a functional chemical model of the manganese cofactor of natural photosynthesis photosystem II

Photogeneration of ozone by marine algae and a functional chemical model of the manganese cofactor of natural photosynthesis photosystem II

Many-electron oxidation of water as treated within the framework of a functional chemical model of the manganese cofactor of oxidase in photosystem II of natural photosynthesis

The kinetics and products of the oxidation of water by MnIV clusters in a 12 M sulfuric acid solution, a functional chemical model of the manganese cofactor of the oxidase in photosystem II of natural photosynthesis, were studied. It was demonstrated that, depending of the conditions, water can be oxidized to either oxygen or ozone. In the reaction mixture, ozone is rapidly and almost completely converted into oxygen. The results obtained were compared to the data on the photosynthetic oxidation of water in red seaweeds.

Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

Iron toxicity and mitochondrial oxidative stress

Manganese containing superoxide dismutase (SOD2) protects mitochondria from oxidative damage, and loss of SOD2 can lead to severe oxidative stress and lethality in various organisms. Enzyme catalysis is carried out by a manganese ion co-factor, and binding of other metals such as iron to the active site would block SOD2 activity. In mitochondria, iron levels are in vast excess over manganese, by nearly 2 orders of magnitude. And SOD2 is predicted to bind iron with the same affinity as manganese. Even so, SOD2 has been reported to bind only its cognate manganese co-factor in vivo. Using the bakers yeast as a model system, we have been probing this apparent exquisite selectivity for manganese over iron. During the course of these studies, we uncovered some surprising aberrations in SOD2 metallation. Specifically, mitochondrial iron overload syndromes can promote iron association with SOD2. Iron precludes manganese from binding to SOD2 in vivo and enzymatic activity is destroyed. Iron also binds to SOD2 when mitochondrial manganese levels are low, even in the absence of iron overload. Our studies have revealed the existence of at least two distinct pools of mitochondrial iron: a SOD2-inert pool that predominates under normal growth conditions and competes poorly with manganese; and a SOD2-reactive form of iron that can become prevalent during iron overload and competes very well with manganese for binding to SOD2. Mitochondrial iron overload has long been associated with oxidative damage, with the primary suspect being iron-catalyzed Fenton chemistry. However, our studies suggest another potentially damaging outcome of iron overload: loss of mitochondrial SOD2. A number of human disorders have been linked to mitochondrial iron overload, and loss of this important anti-oxidant enzyme is a factor worth considering.

Discovery of Protein Phosphatase 2C Inhibitors by Virtual Screening

Protein phosphatase 2C (PP2C) is an archetype of the PPM Ser/Thr phosphatases, characterized by dependence on divalent magnesium or manganese cofactors, absence of known regulatory proteins, and resistance to all known Ser/Thr phosphatase inhibitors. We have used virtual ligand screening with the AutoDock method and the National Cancer Institute Diversity Set to identify small-molecule inhibitors of PP2Calpha activity at a protein substrate. These inhibitors are active in the micromolar range and represent the first non-phosphate-based molecules found to inhibit a type 2C phosphatase. The compounds docked to three recurrent binding sites near the PP2Calpha active site and displayed novel Ser/Thr phosphatase selectivity profiles. Common chemical features of these compounds may form the basis for development of a PP2C inhibitor pharmacophore and may facilitate investigation of PP2C control and cellular function.

A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus.

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.

Manganese activation of superoxide dismutase 2 in Saccharomyces cerevisiae requires MTM1, a member of the mitochondrial carrier family.

Manganese-containing superoxide dismutase (SOD2) plays a critical role in guarding against mitochondrial oxidative stress and is essential for survival of many organisms. Despite the recognized importance of SOD2, nothing is known regarding the mechanisms by which this nuclear-encoded protein is converted to an active enzyme in the mitochondrial matrix. To search for factors that participate in the posttranslational activation of SOD2, we screened for yeast genes that when mutated lead to SOD2 inactivation and identified a single ORF, YGR257c. The encoded protein localizes to the mitochondria and represents a member of the yeast mitochondrial carrier family. YGR257c was previously recognized as the homologue to human CGI-69, a widely expressed mitochondrial carrier family of unknown function. Our studies suggest a connection with SOD2, and we have named the yeast gene MTM1 for manganese trafficking factor for mitochondrial SOD2. Inactivation of yeast MTM1 leads to loss of SOD2 activity that is restored only when cells are treated with high supplements of manganese, but not other heavy metals, indicative of manganese deficiency in the SOD2 polypeptide. Surprisingly, the mitochondrial organelle of mtm1 Delta mutants shows no deficiency in manganese levels. Moreover, mtm1 Delta mutations do not impair activity of a cytosolic version of manganese SOD. We propose that Mtm1p functions in the mitochondrial activation of SOD2 by specifically facilitating insertion of the essential manganese cofactor.