Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data.

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.

Transition Metal Sequestration by the Host-Defense Protein Calprotectin.

In response to microbial infection, the human host deploys metal-sequestering host-defense proteins, which reduce nutrient availability and thereby inhibit microbial growth and virulence. Calprotectin (CP) is an abundant antimicrobial protein released from neutrophils and epithelial cells at sites of infection. CP sequesters divalent first-row transition metal ions to limit the availability of essential metal nutrients in the extracellular space. While functional and clinical studies of CP have been pursued for decades, advances in our understanding of its biological coordination chemistry, which is central to its role in the host-microbe interaction, have been made in more recent years. In this review, we focus on the coordination chemistry of CP and highlight studies of its metal-binding properties and contributions to the metal-withholding innate immune response. Taken together, these recent studies inform our current model of how CP participates in metal homeostasis and immunity, and they provide a foundation for further investigations of a remarkable metal-chelating protein at the host-microbe interface and beyond.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Structural evidence for intermediates during O2 formation in photosystem II.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Photosystem II: the reaction center of oxygenic photosynthesis.

Photosystem II (PSII) uses light energy to split water into chemical products that power the planet. The stripped protons contribute to a membrane electrochemical potential before combining with the stripped electrons to make chemical bonds and releasing O2 for powering respiratory metabolisms. In this review, we provide an overview of the kinetics and thermodynamics of water oxidation that highlights the conserved performance of PSIIs across species. We discuss recent advances in our understanding of the site of water oxidation based upon the improved (1.9-Å resolution) atomic structure of the Mn4CaO5 water-oxidizing complex (WOC) within cyanobacterial PSII. We combine these insights with recent knowledge gained from studies of the biogenesis and assembly of the WOC (called photoassembly) to arrive at a proposed chemical mechanism for water oxidation.

Manganese Increases the Sensitivity of the cGAS-STING Pathway for Double-Stranded DNA and Is Required for the Host Defense against DNA Viruses.

Manganese (Mn) is essential for many physiological processes, but its functions in innate immunity remain undefined. Here, we found that Mn2+ was required for the host defense against DNA viruses by increasing the sensitivity of the DNA sensor cGAS and its downstream adaptor protein STING. Mn2+ was released from membrane-enclosed organelles upon viral infection and accumulated in the cytosol where it bound directly to cGAS. Mn2+ enhanced the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity, enabling cGAS to produce secondary messenger cGAMP in the presence of low concentrations of dsDNA that would otherwise be non-stimulatory. Mn2+ also enhanced STING activity by augmenting cGAMP-STING binding affinity. Mn-deficient mice showed diminished cytokine production and were more vulnerable to DNA viruses, and Mn-deficient STING-deficient mice showed no increased susceptibility. These findings indicate that Mn is critically involved and required for the host defense against DNA viruses.

The Card1 nuclease provides defence during type III CRISPR immunity.

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.

Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase.

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.

PHD2 enzyme is an intracellular manganese sensor that initiates the homeostatic response against elevated manganese.

Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2wild-type. These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis-elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.

Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Bacterial chemolithoautotrophy via manganese oxidation.

Manganese is one of the most abundant elements on Earth. The oxidation of manganese has long been theorized1-yet has not been demonstrated2-4-to fuel the growth of chemolithoautotrophic microorganisms. Here we refine an enrichment culture that exhibits exponential growth dependent on Mn(II) oxidation to a co-culture of two microbial species. Oxidation required viable bacteria at permissive temperatures, which resulted in the generation of small nodules of manganese oxide with which the cells associated. The majority member of the culture-which we designate 'Candidatus Manganitrophus noduliformans'-is affiliated to the phylum Nitrospirae (also known as Nitrospirota), but is distantly related to known species of Nitrospira and Leptospirillum. We isolated the minority member, a betaproteobacterium that does not oxidize Mn(II) alone, and designate it Ramlibacter lithotrophicus. Stable-isotope probing revealed 13CO2 fixation into cellular biomass that was dependent upon Mn(II) oxidation. Transcriptomic analysis revealed candidate pathways for coupling extracellular manganese oxidation to aerobic energy conservation and autotrophic CO2 fixation. These findings expand the known diversity of inorganic metabolisms that support life, and complete a biogeochemical energy cycle for manganese5,6 that may interface with other major global elemental cycles.

Mutation-induced shift of the photosystem II active site reveals insight into conserved water channels.

Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn4CaO5 cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn4CaO5 cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.

Insights into molecular and cellular functions of the Golgi calcium/manganese-proton antiporter TMEM165.

The Golgi compartment performs a number of crucial roles in the cell. However, the exact molecular mechanisms underlying these actions are not fully defined. Pathogenic mutations in genes encoding Golgi proteins may serve as an important source for expanding our knowledge. For instance, mutations in the gene encoding Transmembrane protein 165 (TMEM165) were discovered as a cause of a new type of congenital disorder of glycosylation (CDG). Comprehensive studies of TMEM165 in different model systems, including mammals, yeast, and fish uncovered the new realm of Mn2+ homeostasis regulation. TMEM165 was shown to act as a Ca2+/Mn2+:H+ antiporter in the medial- and trans-Golgi network, pumping the metal ions into the Golgi lumen and protons outside. Disruption of TMEM165 antiporter activity results in defects in N- and O-glycosylation of proteins and glycosylation of lipids. Impaired glycosylation of TMEM165-CDG arises from a lack of Mn2+ within the Golgi. Nevertheless, Mn2+ insufficiency in the Golgi is compensated by the activity of the ATPase SERCA2. TMEM165 turnover has also been found to be regulated by Mn2+ cytosolic concentration. Besides causing CDG, recent investigations have demonstrated the functional involvement of TMEM165 in several other pathologies including cancer and mental health disorders. This systematic review summarizes the available information on TMEM165 molecular structure, cellular function, and its roles in health and disease.

AAV-mediated hepatic expression of SLC30A10 and the Thr95Ile variant attenuates manganese excess and other phenotypes in Slc30a10-deficient mice.

The manganese (Mn) export protein SLC30A10 is essential for Mn excretion via the liver and intestines. Patients with SLC30A10 deficiency develop Mn excess, dystonia, liver disease, and polycythemia. Recent genome-wide association studies revealed a link between the SLC30A10 variant T95I and markers of liver disease. The in vivo relevance of this variant has yet to be investigated. Using in vitro and in vivo models, we explore the impact of the T95I variant on SLC30A10 function. While SLC30A10 I95 expressed at lower levels than T95 in transfected cell lines, both T95 and I95 variants protected cells similarly from Mn-induced toxicity. Adeno-associated virus 8-mediated expression of T95 or I95 SLC30A10 using the liver-specific thyroxine binding globulin promoter normalized liver Mn levels in mice with hepatocyte Slc30a10 deficiency. Furthermore, Adeno-associated virus-mediated expression of T95 or I95 SLC30A10 normalized red blood cell parameters and body weights and attenuated Mn levels and differential gene expression in livers and brains of mice with whole body Slc30a10 deficiency. While our in vivo data do not indicate that the T95I variant significantly compromises SLC30A10 function, it does reinforce the notion that the liver is a key site of SLC30A10 function. It also supports the idea that restoration of hepatic SLC30A10 expression is sufficient to attenuate phenotypes in SLC30A10 deficiency.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis.

Manganese-dependent transcription regulation by MntR and PerR in Thermus thermophilus HB8.

Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA-binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA-binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies in Thermus thermophilus HB8. By homology search, we classify the DtxR homolog as a manganese-specific, MntR (TtMntR), and the FUR homolog as a peroxide-sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes in vivo, and TtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We show TtPerR and TtMntR bind DNA in the presence of manganese in vitro and in vivo; however, TtPerR is unable to bind DNA in the presence of iron, likely due to iron-mediated histidine oxidation. Unlike canonical PerR homologs, TtPerR does not appear to contribute to peroxide detoxification. Instead, the TtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

Zinc and manganese homeostasis closely interact in mammalian cells.

Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn-related diseases in the future.

Light-driven anaerobic microbial oxidation of manganese.

Oxygenic photosynthesis supplies organic carbon to the modern biosphere, but it is uncertain when this metabolism originated. It has previously been proposed1,2 that photosynthetic reaction centres capable of splitting water arose by about 3 billion years ago on the basis of the inferred presence of manganese oxides in Archaean sedimentary rocks. However, this assumes that manganese oxides can be produced only in the presence of molecular oxygen3, reactive oxygen species4,5 or by high-potential photosynthetic reaction centres6,7. Here we show that communities of anoxygenic photosynthetic microorganisms biomineralize manganese oxides in the absence of molecular oxygen and high-potential photosynthetic reaction centres. Microbial oxidation of Mn(II) under strictly anaerobic conditions during the Archaean eon would have produced geochemical signals identical to those used to date the evolution of oxygenic photosynthesis before the Great Oxidation Event1,2. This light-dependent process may also produce manganese oxides in the photic zones of modern anoxic water bodies and sediments.

A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin.

The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)-the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series-is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors' selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.