Deciphering the Catalytic Mechanism of Human Manganese Superoxide Dismutase

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of human SODs that dwells within the mitochondria and is responsible for protecting the organelle against superoxide-mediated damage. The protein is arguably the most significant antioxidant enzyme as the mitochondria are especially integral for cellular vitality. This is exemplified by the embryonic lethality of mice lacking MnSOD and the multitude of human disease states that manifest as a result of dysfunctional MnSOD. The bioprotective attributes of MnSOD have attracted the attention of clinicians and is illustrated by the multiple ongoing clinical trials that attempt to mimic the function of the enzyme. While MnSOD has proven to be of significant importance for human vitality and has been studied extensively since its discovery over 50 years ago, its atom-by-atom mechanism has still been elusive and the mechanism of MnSOD has yet to be defined due to its nature of catalysis. MnSOD performs its function through concerted proton-electron transfers (CPETs) at specific sites of the enzyme that have been extremely difficult to detect experimentally. An emerging biophysical tool capable of circumventing previous experimental obstacles is neutron protein crystallography. This method involves diffracting neutrons off of crystallized protein samples with controlled electronic states into a pattern that can be deciphered for specific proton sites thereby permitting the experimental coupling of proton and electron transfers. In this thesis work, significant revelations are made for the mechanism of MnSOD using a multitude of approaches, including neutron crystallography where significant developments are also made for the emerging technique

A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ~1% of the catalytic activity of the recombinant wild-type enzyme, and have altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ~11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz \u3c gx,y. 14N-hyperfine is observed on gz, confirming the addition of the apical histidine ligand in the Ni(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models

Metal specificities and catalytic activities of the two superoxide dismutases of Staphylococcus aureus

PhD ThesisThe superoxide dismutase (SOD) metalloenzymes play a major role in the cellular oxidative stress defence systems of microorganisms including that of an important pathogenic bacterium Staphylococcus aureus. Staphylococcus aureus, unlike other staphylococci, possesses two isozymes of the Fe/Mn-dependent SOD superfamily, designated SodA and SodM, both of which are predicted to utilise manganese as their essential metal cofactor. The two SODs are critical for the resistance to reactive oxygen species (ROS) and contribute to the pathogenicity of S. aureus. The immune system can utilise a Mn-restriction as one of its defence mechanism against pathogens, suggesting a possibility that one of the S. aureus SODs can use another metal in order to overcome this host-imposed Mn-starvation. To clarify the metal requirements of the two S. aureus SODs, the recombinant Fe- and Mnmetalated isoforms of S. aureus SodA and SodM were produced. All four forms were characterised as catalytically active, regardless of utilised metal. The relative activity analysis showed that SodA exhibits a strong metal preference of Mn over Fe, whereas SodM presented highly cambialistic properties, i.e. it was equally active with either Mn or Fe. Crystal structures of all four forms of the S. aureus SOD proteins were solved and showed a high level of identity. Structure-based mutagenesis led to the successful swapping of catalytic properties between the two proteins, yielding a Mn-specific SodM and a cambialistic SodA, with no significant change to an overall enzyme architecture. HF-EPR analysis gave insight into the mechanism of metal-specific catalysis of the two enzymes. Phylogenetic analyses suggested the cambialistic SodM originated from a gene duplication of a single, likely Mn-specific SOD, in the common ancestor of all analysed S. aureus isolates. The evolution of SodM to work with both metals could have provided an important adaptation for resisting manganese-starvation during infection. Characterisation of SodM purified directly from S. aureus, as well as studies in an animal model of infection, provided evidence consistent with the hypothesis that the cambialistic SodM contributes to resisting host-imposed metal starvation during S. aureus infection.BBSRC as part of Newcastle-Liverpool-Durham Doctoral Training Partnership

Manganese biogeochemistry in the sunlit ocean = Die Biogeochemie des Mangans in der euphotischen Zone

The trace metal manganese (Mn) plays a significant role in seawater as it is bio-essential for phytoplankton. Mn plays a critical role as a redox center in Photosystem II (PSII) during the conversion of water to oxygen in photosynthesis. It is also essential in other redox related enzymatic processes; in particular Mn is important as the active metal center in superoxide dismutase (SOD) which provides intracellular protection against oxidative stress due to photochemically produced superoxide (O2 ). Mn exists in seawater in three redox states: soluble and prevalent Mn(II), insoluble Mn(III) and Mn(IV)-oxides. In the euphotic zone the biogeochemical cycling of Mn is strongly influenced by reactive oxygen species (ROS). The highly reactive and short-lived superoxide (O2 ) and hydrogen peroxide (H2O2) can both act as oxidants and reductants, and they play a key role in the Mn processes in seawater. For example the dominant Mn sources to the open ocean are the Mn-oxides which are present in atmospheric dust which are reduced to soluble Mn(II) by photochemically produced H2O2. While these processes have been crudely identified, the dominant reactions and mechanisms of Mn and ROS in seawater are poorly understood. This lack of knowledge demands investigations into the in-situ dissolution processes of Mn from dust and into studying the exact reaction mechanisms between Mn and ROS in the euphotic zone. This thesis comprises four manuscripts. Manuscripts 1 and 2 (Wuttig et al., subm., 2013a; Wuttig et al., subm., 2013b) focus on the cycling and reaction mechanisms of Mn and ROS. Manuscript 3 (Wuttig et al., in prep., 2013) addresses differences in the input and distribution of cadmium (Cd), iron (Fe) and Mn in the Eastern Tropical Atlantic Ocean off Cape Verde, and manuscript 4 (Wuttig et al., 2013) describes Mn cycling after dust additions in a trace metal clean mesocosm experiment in the Mediterranean Sea. This study has conclusively shown that Mn and organic matter are the dominant sinks for O2 in the Eastern Tropical North Atlantic (manuscripts 1; Wuttig et al., subm., 2013a). Mn dominates this decay especially in the surface waters which are influenced by high atmospheric dust deposition and near the sediment/water interface due to Mn sediment resuspension. This contrasts with current knowledge based on findings from the Mn poor Southern Ocean where copper (Cu) was shown to be the major sink. In manuscript 2 it is demonstrated that O2 decays by reaction with inorganic Mn(II) in seawater following a first order loss rate which appears to involve a catalytic reaction involving the Mn(II)/MnO2+ couple, in which MnO2+ is a manganous superoxide complex (Wuttig et al., subm., 2013a). Thus in sunlit and oxygenated waters Mn(III) is unlikely to be found in significant concentrations when strong Mn(III) binding ligands are not present. In other studies Mn(III) was found under anoxic conditions in the presence of unknown strong Mn(III) binding ligands. Therefore, in contrast to the Mn(II)/MnO2+ pair, Mn(III) cannot act as a SOD in the oxygenated surface ocean. In the Eastern Tropical North Atlantic Ocean atmospheric dust is the main source of Mn to surface waters (manuscript 3; Wuttig et al., in prep., 2013). However this study provides clear evidence that equatorial upwelling and sediment resuspension are important Mn sources in this region. In contrast to findings from the Eastern Tropical Pacific, where unexpected high surface concentrations were observed, no secondary Mn(II) maximum was found in the Eastern Tropical North Atlantic Ocean. This could have been introduced by a combination of lateral transport of Mn rich waters from the coastal margins and reduction of Mn-oxides. While Aeolian sources were predominantly influencing Mn and also Fe cycling in the Eastern Tropical Atlantic, Cd was not controlled by dust deposition (manuscript 3; Wuttig et al., in prep., 2013). These biologically relevant elements exhibited contrasting distribution patterns. For Fe and Mn, atmospheric depositions masked a classical nutrient type profile, while Cd was very depleted at the surface and concentrations steadily increased with depth. Cd was highly correlated to Phosphate (hereafter referred to as P). The Cd/P ratio was mainly controlled by P with elevated concentrations at depth resulting in strongly differing ratios in surface and subsurface layers of 16.6 pmol / µmol and 237 pmol / µmol, respectively. The complex photochemical processes during the dissolution of Mn dust are also subject of manuscript 4. This paper describes a mesocosm project in the Mediterranean with two consecutive additions of evapocondensed dust conducted. The data also show that the dissolution and loss rates of Mn were comparable during both seedings. The calculated fractional solubilities for the first and the second dust addition were 41 ± 9 % and 27 ± 19 %, respectively. The results presented in this thesis have significantly improved our understanding of Mn distribution and especially cycling in the euphotic zone. An insight into the mechanisms between Mn and ROS and into the dissolution processes from dust is given

Manganese superoxide dismutase from Thermus thermophilus: A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor (= [summation operator]||Fo|-|Fc||/[summation operator]|Fo|) for the 54,056 unique reflections measured between 10[middle dot]0 and 1[middle dot]8 A (96% of all possible reflections) is 0[middle dot]176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P41212 cell.The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an [alpha] + [beta] structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the [beta]-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions.The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/ monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain.Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal. A pocket adjoining the metal, formed by His33, Trp87, His83 and Tyr36, is postulated to be the substrate-binding site. Refinement of 2.3 A data from crystals reduced with dithionite indicates that the co-ordination geometry at the metal is not changed by reduction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29328/1/0000395.pd

Study of the Role of Biologically-Relevant, Labile Nickel Pools in the Maturation of Nickel-Dependent Enzymes

Cellular nickel pools, comprised of static and labile pools of nickel complexes, play important roles in maintaining nickel homeostasis in various organisms (microbes, fungi, and plants), which utilize it as a cofactor of one or more nickel enzymes that catalyze specific reactions and are essential for their proper growth and survival in various ecological niches. Like other metals, tight regulation of cellular nickel levels is critical to prevent toxic effects of nickel deprivation, nickel overload, and ‘free’ nickel. While more static nickel pools include nickel tightly bound to nickel-dependent enzymes, nickel in the labile pool is exchangeable and weakly bound to either nickel chaperones or low-molecular-weight (LMW) ligands, as is the case for many other transition metals. The role of nickel chaperones in enzyme maturation and activation is being extensively investigated, but the importance of cellular LMW complexes in the process remains largely unknown. In this work, I investigated the role of labile nickel pools (both non-proteinaceous and proteinaceous ligands) in the maturation of nickel-dependent enzymes - Nickel Superoxide Dismutase (NiSOD) from Streptomyces coelicolor, and urease and Ni, Fe-hydrogenase from Helicobacter pylori. For the maturation of NiSOD, no chaperone has been characterized for nickel delivery to the active site. Using biochemical assays, mass spectrometry, and spectroscopic methods, I provide compelling evidence that biologically relevant, non-proteinaceous, LMW ligand complexes of nickel with L-histidine are inevitable for the proper maturation of the enzyme. In Helicobacter pylori, which is a human pathogen, there are two nickel dependent enzymes – urease and Ni, Fe hydrogenase both of which require a proteinaceous labile nickel ligand (common nickel-chaperone), HypA for maturation. HypA interacts with HypB for the maturation of Ni, Fe-hydrogenase, and with UreE for the maturation of urease, but what factors determine its differential interaction with its downstream partner proteins are unknown. I interrogated the flexibility and dynamics of HypA by glycine mutations to perturb the maturation of the two nickel enzymes in the pathogen and using in vitro assays I show that the glycine mutations differentially affect the maturation of urease and Ni, Fe – hydrogenase in the pathogen

The Effects of Ionizing Radiation and Oxidizing Species on Strains of Deinococcus radiodurans Lacking Endogenous Oxidative Protection Methods

Multiple strains of Deinococcus radiodurans were transformed, creating knockout mutations in genes responsible for manganese ion transport, manganese and copper/zinc super-oxide dismutase, and bacillithiol synthesis. These mutated strains were then irradiated with ~20,000 Gys. The results showed that the mutated strains had a higher sensitivity to ionizing radiation, those responsible for bacillithiol synthesis having an increase in sensitivity 3000 times more than wild type Deinococcus radiodurans. In addition to radiation the mutated strains were also exposed to paraquat, an oxidizing herbicide. Strains missing manganese super-oxide dismutase showed increased sensitivity

Iron–Nutrient Interactions within Phytoplankton

Iron limits photosynthetic activity in up to one third of the world’s oceans and in many fresh water environments. When studying the effects of Fe limitation on phytoplankton or their adaptation to low Fe environments, we must take into account the numerous cellular processes within which this micronutrient plays a central role. Due to its flexible redox chemistry, Fe is indispensable in enzymatic catalysis and electron transfer reactions and is therefore closely linked to the acquisition, assimilation and utilization of essential resources. Iron limitation will therefore influence a wide range of metabolic pathways within phytoplankton, most prominently photosynthesis. In this review we map out four well-studied interactions between Fe and essential resources: nitrogen, manganese, copper and light. Data was compiled from both field and laboratory studies to shed light on larger scale questions such as the connection between metabolic pathways and ambient iron levels and the biogeographical distribution of phytoplankton species