Redox thermodynamics of B-class dye-decolorizing peroxidases

With>5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors. To understand these characteristics, we have investigated the redox thermodynamics of the one-electron reduction of the ferric high-spin form of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) and the variants D143A, R232A and D143A/R232A. These distal amino acids are fully conserved in all DyPs and play important roles in Compound I formation and maintenance of the heme cavity architecture and substrate access route(s). The E°′ values of the respective redox couples Fe(III)/Fe(II) varied from −350 mV (wild-type KpDyP) to −299 mV (D143A/R232A) at pH 7.0. Variable-temperature spectroelectrochemical experiments revealed that the reduction reaction of B-class DyPs is enthalpically unfavored but entropically favored with significant differences in enthalpic and entropic contributions to E°′ between the four proteins. Molecular dynamics simulations demonstrated the impact of solvent reorganization on the entropy change during reduction reaction and revealed the dynamics and restriction of substrate access channels. Obtained data are discussed with respect to the poor peroxidase activities of B-class DyPs and compared with heme peroxidases from other (super)families as well as with chlorite dismutases, which do not react with hydrogen peroxide but share a similar fold and heme cavity architecture

J. Mol. Biol.

Cyanobacteria are shown to be unique in containing membrane- bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this Phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0 Angstrom resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OHx) a five-coordinated trigonal bipyramidal geometry is formed, with OHx and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OHx. The ligands including OHx are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn...Mn distance (18.4Angstrom) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented. (C) 2002 Elsevier Science Ltd. All rights reserved

The 2.0 angstrom resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane- bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this Phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0 Angstrom resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OHx) a five-coordinated trigonal bipyramidal geometry is formed, with OHx and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OHx. The ligands including OHx are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn...Mn distance (18.4Angstrom) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented. (C) 2002 Elsevier Science Ltd. All rights reserved

Biochemical characterization of a membrane-bound manganese- containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta28)) or the hydrophobic and the linker region (MnSOD(Delta60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg(-1) protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta28)) or (MnSOD(Delta60)) concentrations is first- order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 106 M-1 s(-1) (pH 10) and 6 X 10(7) M-1 S-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria

Redox thermodynamics of the Fe3+/Fe2+ couple in wild type and mutated heme peroxidases

The thermodynamics of the one-electron reduction of the ferricheme in wild-type and mutated heme Synechocystis catalaseperoxidase and human myeloperoxidase were determined through spectro-electrochemical experiments. The data are interpreted in terms of ligand binding features, electrostatic effects and solvation properties of the heme environment

Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionatesubstituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (-50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidationstates showed significant differences. Additionally, rates of ligand binding and oxidation of both one and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His-Asn-Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate bindingsite(s) at the entrance to the heme cavity is discussed