R2-like Ligand-binding Oxidase Research Articles

Site-Differentiated MnIIFeII Complex Reproducing the Selective Assembly of Biological Heterobimetallic Mn/Fe Cofactors.

Class Ic ribonucleotide reductases (RNRIc) and R2-like ligand-binding oxidases (R2lox) are known to contain heterobimetallic MnIIFeII cofactors. How these enzymes assemble MnIIFeII cofactors has been a long-standing puzzle due to the weaker binding affinity of MnII versus FeII. In addition, the heterobimetallic selectivity of RNRIc and R2lox has yet to be reproduced with coordination complexes, leading to the hypothesis that RNRIc and R2lox overcome the thermodynamic preference for coordination of FeII over MnII with their carefully constructed three-dimensional protein structures. Herein, we report the selective formation of a heterobimetallic MnIIFeII complex accomplished in the absence of a protein scaffold. Treatment of the ligand Py4DMcT (L) with equimolar amounts of FeII and MnII along with two equivalents of acetate (OAc) affords [LMnIIFeII (OAc)2(OTf)]+ (MnIIFeII) in 80% yield, while the diiron complex [LFeIIFeII(OAc)2(OTf)]+ (FeIIFeII) is produced in only 8% yield. The formation of MnIIFeII is favored regardless of the order of addition of FeII and MnII sources. X-ray diffraction (XRD) of single crystals of MnIIFeII reveals an unsymmetrically coordinated carboxylate ligand─a primary coordination sphere feature shared by both RNRIc and R2lox that differentiates the two metal binding sites. Anomalous XRD studies confirm that MnIIFeII exhibits the same site selectivity as R2lox and RNRIc, with the FeII (d6) center preferentially occupying the distorted octahedral site. We conclude that the successful assembly of MnIIFeII originates from (1) Fe-deficient conditions, (2) site differentiation, and (3) the inability of ligand L to house a dimanganese complex.

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A MnIII(μ-O)(μ-OH)FeIII species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(μ-O)(μ-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded MnIII(μ-OH)FeIII species. This final intermediate precedes formation of the MnIII/FeIII resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.

Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

R2‐like ligand‐binding oxidase (R2lox) is a ferritin‐like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross‐link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry‐based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

Spontaneous Formation of an Fe/Mn Diamond Core: Models for the Fe/Mn Sites in Class 1c Ribonucleotide Reductases.

A handful of oxygen-activating enzymes has recently been found to contain Fe/Mn active sites, like Class 1c ribonucleotide reductases and R2-like ligand-binding oxidase, which are closely related to their better characterized diiron cousins. These enzymes are proposed to form high-valent intermediates with Fe-O-Mn cores. Herein, we report the first examples of synthetic Fe/Mn complexes that mimic doubly bridged intermediates proposed for enzymatic oxygen activation. Fe K-edge extended X-ray absorption fine structure (EXAFS) analysis has been used to characterize the structures of each of these compounds. Linear compounds accurately model the Fe···Mn distances found in Fe/Mn proteins in their resting states, and doubly bridged diamond core compounds accurately model the distances found in high-valent biological intermediates. Unlike their diiron analogues, the paramagnetic nature of Fe/Mn compounds can be analyzed by EPR, revealing S = 1/2 signals that reflect antiferromagnetic coupling between the high-spin Fe(III) and Mn(III) units of heterobimetallic centers. These compounds undergo electron transfer with various ferrocenes, linear compounds being capable of oxidizing diacetyl ferrocene, a weak reductant, and diamond core compounds being capable of oxidizing acetyl ferrocene. Diamond core compounds can also perform HAT reactions from substrates with X-H bonds with bond dissociation free energies (BDFEs) up to 75 kcal/mol and are capable of oxidizing TEMPO-H at rates of 0.32-0.37 M-1 s-1, which are comparable to those reported for some mononuclear FeIII-OH and MnIII-OH compounds. However, such reactivity is not observed for the corresponding diiron compounds, a difference that Nature may have taken advantage of in evolving enzymes with heterobimetallic active sites.

Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein.

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

Structure of a novel R2-like ligand-binding oxidase revealed by electron diffraction

Structure of a novel R2-like ligand-binding oxidase revealed by electron diffraction

Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD

Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine\u2013valine ether cross-link formation in the R2-like ligand-binding oxidase

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine–valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.

Ether cross-link formation in the R2-like ligand-binding oxidase

R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine–valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

Oxoiron(IV) complexes as synthons for the assembly of heterobimetallic centers such as the Fe/Mn active site of Class Ic ribonucleotide reductases.

Nonheme oxoiron(IV) complexes can serve as synthons for generating heterobimetallic oxo-bridged dimetal complexes by reaction with divalent metal complexes. The formation of FeIII-O-CrIII and FeIII-O-MnIII complexes is described herein. The latter complexes may serve as models for the FeIII-X-MnIII active sites of an emerging class of Fe/Mn enzymes represented by the Class 1c ribonucleotide reductase from Chlamydia trachomatis and the R2-like ligand-binding oxidase (R2lox) found in Mycobacterium tuberculosis. These synthetic complexes have been characterized by UV-Vis, resonance Raman, and X-ray absorption spectroscopy, as well as electrospray mass spectrometry. The FeIII-O-CrIII complexes exhibit a three-band UV-Vis pattern that differs from the simpler features associated with FeIII-O-FeIII complexes. The positions of these features are modulated by the nature of the supporting polydentate ligand on the iron center, and their bands intensify dramatically in two examples upon the binding of an axial cyanate or thiocyanate ligand trans to the oxo bridge. In contrast, the FeIII-O-MnIII complexes resemble FeIII-O-FeIII complexes more closely. Resonance Raman characterization of the FeIII-O-MIII complexes reveals an 18O-sensitive vibration in the range of 760-890cm-1. This feature has been assigned to the asymmetric FeIII-O-MIII stretching mode and correlates reasonably with the Fe-O bond distance determined by EXAFS analysis. The likely binding of an acetate as a bridging ligand to the FeIII-O-MnIII complex 12 lays the foundation for further efforts to model the heterobimetallic active sites of Fe/Mn enzymes.

Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of MnII and FeII. Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess MnII promotes heterobimetallic cofactor assembly. In the absence of FeII, R2c cooperatively binds MnII at both metal sites, whereas R2lox does not readily bind MnII at either site. Heterometallic cofactor assembly is favored at substoichiometric FeII concentrations in R2lox. FeII and MnII likely bind to the protein in a stepwise fashion, with FeII binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of MnII by FeII at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular MnII/FeII concentrations in the host organisms from which they were isolated.

Enhanced Stability of the FeII/MnII State in a Synthetic Model of Heterobimetallic Cofactor Assembly

Heterobimetallic Mn/Fe cofactors are found in the R2 subunit of class Ic ribonucleotide reductases (R2c) and R2-like ligand binding oxidases (R2lox). Selective cofactor assembly is due at least in part to the thermodynamics of M(II) binding to the apoprotein. We report here equilibrium studies of Fe(II)/Mn(II) discrimination in the biomimetic model system H5(F-HXTA) (5-fluoro-2-hydroxy-1,3-xylene-α,α'-diamine-N,N,N',N'-tetraacetic acid). The homobimetallic F-HXTA complexes [Fe(H2O)6][1]2·14H2O and [Mn(H2O)6][2]2·14H2O (1 = [Fe(II)2(F-HXTA)(H2O)4](-); 2 = [Mn(II)2(F-HXTA)(H2O)4](-)) were characterized by single crystal X-ray diffraction. NMR data show that 1 retains its structure in solution (2 is NMR silent). Metal exchange is facile, and the heterobimetallic complex [Fe(II)Mn(II)(F-HXTA)(H2O)4](-) (3) is formed from mixtures of 1 and 2. (19)F NMR was used to quantify 1 and 3 in the presence of excess M(II)(aq) at various metal ratios, and equilibrium constants for Fe(II)/Mn(II) discrimination were calculated from these data. Fe(II) is preferred over Mn(II) with K1 = 182 ± 13 for complete replacement (2 ⇌ 1). This relatively modest preference is attributed to a hard-soft acid-base mismatch between the divalent cations and the polycarboxylate ligand. The stepwise constants for replacement are K2 = 20.1 ± 1.3 (2 ⇌ 3) and K3 = 9.1 ± 1.1 (3 ⇌ 1). K2 > K3 demonstrates enhanced stability of the heterobimetallic state beyond what is expected for simple Mn(II) → Fe(II) replacement. The relevance to Fe(II)/Mn(II) discrimination in R2c and R2lox proteins is discussed.

Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor

Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins.

The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.

A Dynamic C‐Terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein Can Adopt a Helical Structure with Possible Functional Consequences

Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.