An Active Dimanganese(III)−Tyrosyl Radical Cofactor in <i>Escherichia coli</i> Class Ib Ribonucleotide Reductase

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: α2 (NrdE), where nucleotide reduction occurs, and β2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)6-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with MnII and incubated with fully reduced NrdI (NrdIhq) and O2. Active RNR was rapidly produced with 0.25 ± 0.03 tyrosyl radical (Y·) per β2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is MnIII2-Y·, which we propose is generated by MnII2-NrdF reacting with two equivalents of HO2−, produced by reduction of O2 by NrdF-bound NrdIhq. In the absence of NrdIhq, with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with FeII and incubated with O2 in the presence or absence of NrdIhq gave 0.2 and 0.7 Y·/β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdIhq hinders FeIII2-Y· cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the MnIII2-Y· cofactor, not the diferric-Y· one, is the active metallocofactor in vivo

The <i>czcD</i> (NiCo) Riboswitch Responds to Iron(II)

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to FeII. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to NiII and CoII but thought not to respond to FeII, on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to FeII, as well as to MnII, CoII, NiII, and ZnII, but only in the cases of FeII and MnII do the apparent Kd values (0.4 and 11 μM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensorwhich is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for FeIIresponds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present

<i>Escherichia coli</i> Class Ib Ribonucleotide Reductase Contains a Dimanganese(III)-Tyrosyl Radical Cofactor in Vivo

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (FeIII2-Y•) and dimanganese(III)-Y• (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297−1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ∼95% purity, with 0.2 Y•/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min−1 mg−1. Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo

Reconsidering the <i>czcD</i> (NiCo) Riboswitch as an Iron Riboswitch

Recent work has proposed a new mechanism of bacterial iron regulation: riboswitches that undergo a conformational change in response to FeII. The czcD (NiCo) riboswitch was initially proposed to be specific for NiII and CoII, but we recently showed via a czcD-based fluorescent sensor that FeII is also a plausible physiological ligand for this riboswitch class. Here, we provide direct evidence that this riboswitch class responds to FeII. Isothermal titration calorimetry studies of the native czcD riboswitches from three organisms show no response to MnII, a weak response to ZnII, and similar dissociation constants (∼1 μM) and conformational responses for FeII, CoII, and NiII. Only the iron response is in the physiological concentration regime; the riboswitches’ responses to CoII, NiII, and ZnII require 103-, 105-, and 106-fold higher “free” metal ion concentrations, respectively, than the typical availability of those metal ions in cells. By contrast, the “Sensei” RNA, recently claimed to be an iron-specific riboswitch, exhibits no response to FeII. Our results demonstrate that iron responsiveness is a conserved property of czcD riboswitches and clarify that this is the only family of iron-responsive riboswitch identified to date, setting the stage for characterization of their physiological function

A Periplasmic Binding Protein for Pyrroloquinoline Quinone

Pyrroloquinoline quinone (PQQ) is an essential redox cofactor in bacterial calcium- and lanthanide-dependent alcohol dehydrogenases. Although certain bacteria are known to synthesize and secrete PQQ, little is known about trafficking of this cofactor within and between cells. Here, we show that a previously uncharacterized periplasmic (solute) binding protein from Methylobacterium extorquens AM1, here renamed PqqT, binds 1 equiv of PQQ with high affinity (Kd = 50 nM). UV–visible and spectrofluorometric titrations establish that PqqT binds an unhydrated form of PQQ with distinct spectral features from the cofactor in free solution. To our knowledge, PqqT is the first solute-binding protein identified for PQQ and the first protein implicated in cellular trafficking of the cofactor. We propose that PqqT, which is encoded adjacent to a putative ATP-binding cassette transporter in the M. extorquens genome, is involved in uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis. These results support the emerging importance of PQQ transfer within microbial and microbe–host communities

A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements

Sensitive yet rapid methods for detection of rare earth elements (REEs), including lanthanides (Lns), would facilitate mining and recycling of these elements. Here we report a highly selective, genetically encoded fluorescent sensor for Lns, LaMP1, based on the recently characterized protein, lanmodulin. LaMP1 displays a 7-fold ratiometric response to all LnIIIs, with apparent Kds of 10–50 pM but only weak response to other common divalent and trivalent metal ions. We use LaMP1 to demonstrate for the first time that a Ln-utilizing bacterium, Methylobacterium extorquens, selectively transports early Lns (LaIII–NdIII) into its cytosol, a surprising observation as the only Ln-proteins identified to date are periplasmic. Finally, we apply LaMP1 to suggest the existence of a LnIII uptake system utilizing a secreted metal chelator, akin to siderophore-mediated FeIII acquisition. LaMP1 not only sheds light on Ln biology but also may be a useful technology for detecting and quantifying REEs in environmental and industrial samples

Probing Lanmodulin’s Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage

Lanmodulin is the first natural, selective macrochelator for f elementsa protein that binds lanthanides with picomolar affinity at 3 EF hands, motifs that instead bind calcium in most other proteins. Here, we use sensitized terbium luminescence to probe the mechanism of lanthanide recognition by this protein as well as to develop a terbium-specific biosensor that can be applied directly in environmental samples. By incorporating tryptophan residues into specific EF hands, we infer the order of metal binding of these three sites. Despite lanmodulin’s remarkable lanthanide binding properties, its coordination of approximately two solvent molecules per site (by luminescence lifetime) and metal dissociation kinetics (koff = 0.02–0.05 s–1, by stopped-flow fluorescence) are revealed to be rather ordinary among EF hands; what sets lanmodulin apart is that metal association is nearly diffusion limited (kon ≈ 109 M–1 s–1). Finally, we show that Trp-substituted lanmodulin can quantify 3 ppb (18 nM) terbium directly in acid mine drainage at pH 3.2 in the presence of a 100-fold excess of other rare earths and a 100 000-fold excess of other metals using a plate reader. These studies not only yield insight into lanmodulin’s mechanism of lanthanide recognition and the structures of its metal binding sites but also show that this protein’s unique combination of affinity and selectivity outperforms synthetic luminescence-based sensors, opening the door to rapid and inexpensive methods for selective sensing of individual lanthanides in the environment and in-line monitoring in industrial operations

Structural Basis for Rare Earth Element Recognition by <i>Methylobacterium extorquens</i> Lanmodulin

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with CaII binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for LnIIIs and YIII over CaII. Here we present the nuclear magnetic resonance solution structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein’s picomolar affinity for LnIIIs, and it suggests a role of unusual Ni+1–H···Ni hydrogen bonds, in which LanM’s unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM’s Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals

Lanmodulin’s EF 2–3 Domain: Insights from Infrared Spectroscopy and Simulations

Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2–3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from Methylorubrum extorquens. Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2–3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants (Kd) in the range 20–30 μM, with no significant differences in the Kd values for La3+, Eu3+, and Tb3+ at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2–3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2–3 with all three lanthanides. MD simulations, supported by Eu3+ luminescence measurements, explore these results, suggesting a competition between water–lanthanide and carboxylate–lanthanide interactions in the EF 2–3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein