Differentiated, promoter-specific response of [4Fe-4S] NsrR DNA-binding to reaction with nitric oxide

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA-binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ~2 NO per cluster, while for the hmpA1 and nsrR promoters, ~4 and ~8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8 – 10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ~2 NO per cluster, along with two further intermediates at ~4 and ~6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA-binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn

Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+ via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2O2, DyPs form a high‐valent iron(IV)‐oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein‐bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein‐bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron‐transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron‐transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed

Engineering tyrosine-based electron flow pathways in proteins: The case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed. © 2012 American Chemical Society

Heterogeneity in the histidine-brace copper coordination sphere in auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E- Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell

Electron transfer from haem to the di-iron ferroxidase centre in bacterioferritin

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins

Naturally Occurring Disease-Related Mutations in the 40–57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization

American Chemical Society. Naturally occurring mutations found in one of the two ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (pKtrigger). A second deprotonation stabilizes a pentacoordinate heme form (pKa2). In this study, site-directed variants at the 41 and 48 positions in the 40-57 ω-loop and at the 81 and 83 positions in the 71-85 ω-loop reveal that conformational transitions in the 71-85 ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the pKtriggerand increase pKa2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength

Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis

Reaction of O2 with a di-iron protein generates a mixed valent Fe2+/Fe3+ center and peroxide

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron–O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions