Julia J. Griese

Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors: R2lox and R2c Proteins

The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Significance Metallocofactors enable enzymes to catalyze difficult reactions that would otherwise not be possible, such as the reduction of oxygen. Nature utilizes a number of different metals, and it is crucial that proteins bind the correct metals to execute their function. Nonetheless, the principles that govern metal specificity in proteins remain poorly understood. Here we use an enzyme that forms a heterodinuclear Mn/Fe cofactor with the same protein ligands in both metal-coordinating positions to study how proteins can differentiate between two such similar metals. We show that the protein is intrinsically capable of site-specific metal discrimination. Surprisingly, specificity is achieved in a stepwise process involving not only fundamental affinity differences, but also chemical maturation upon reaction with molecular oxygen. Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving–Williams series (MnII < FeII < NiII < CoII < CuII > ZnII). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving–Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine–valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

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.

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

Background: R2-like ligand-binding oxidases (R2lox) can assemble a Mn/Fe or diiron cofactor. Results: The metal centers are structurally similar and activate oxygen, resulting in redox-coupled structural changes. Conclusion: Oxygen activation likely proceeds via similar mechanisms at Mn/Fe and diiron clusters, while their redox state controls oxygen and substrate access. Significance: R2lox proteins could provide novel catalysts for oxidative chemistry. 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 MnII and FeII 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.

Characterization of Oxygen Bridged Manganese Model Complexes Using Multifrequency (17)O-Hyperfine EPR Spectroscopies and Density Functional Theory.

Multifrequency pulsed EPR data are reported for a series of oxygen bridged (μ-oxo/μ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-μ-oxo bridged complex and a bent, bis-μ-oxo-μ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the μ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.

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.

X-ray reduction correlates with soaking accessibility as judged from four non-crystallographically related diiron sites

X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.

Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase

The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine cross-link near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Φ = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligand-to-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

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.