Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin.

The cleavage of the axial S(Met)-Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV-VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe−M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467–487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe-S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt specie

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria, in 2015 by Dailey and coworkers triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely H182A and E263Q, involving two key active site residues. Interestingly, both variants are active only towards the physiological substrate coproporphyrin III but inactive towards protoporphyrin IX. In addition, E263 is impairing the final oxidation from ferrous coproheme to ferric coproheme. The characteristics of the active site in terms of the residues involved and the substrate binding properties are discussed by structural and functional means, providing a further contribution to the deciphering of the enigmatic reaction mechanism

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

Met80 and Tyr67 affect the chemical unfolding of yeast cytochrome c: comparing solution vs. immobilized state

Urea-induced denaturation of the Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) was studied through variable temperature diffusive cyclic voltammetry and electronic absorption, CD and MCD spectroscopies. The susceptibility to unfolding of both variants - represented by the free energy of unfolding at denaturant infinite dilution, ∆〖G°〗_u^(H_2 O)is greater compared to the species showing an intact Met/His coordination, as observed previously for the same species immobilized onto a functionalized electrode. This is consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Notably, we find that the unfolding propensity and axial heme iron coordination of the present Fe-(S)Met bond-deprived variants is affected by the motional regime of the protein. In particular, electrostatic adsorption onto a negatively charged SAM surface - that would mimic the phospholipidic inner mitochondrial membrane - facilitates unfolding compared to the solution state, especially at room temperature. This finding has a physiological relevance related to the cytochrome c interaction with cardiolipin at the IMM in the early stages of apoptosis. Moreover, while both immobilized variants maintain the His/OH- axial heme iron coordination up to 7 M urea, the same species in solution are subjected to urea-induced replacement of the axial hydroxide ligand by a His ligand. The contribution of the enthalpic and entropic terms to ∆〖G°〗_u^(H_2 O) were found to be opposite (H-S compensation) indicating that unfolding thermodynamics are strongly affected by changes in the hydrogen bonding network in the hydration sphere of the protein

Compound I formation and reactivity in dimeric chlorite dismutase – Impact of pH and the dynamics of the catalytic arginine

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation

Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward \u3b2-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates

Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) may 34 contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite “dismutase”, Cld). Beside the water-splitting manganese complex of photosystem II this metalloenzyme is the second known enzyme that catalyzes the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in E. coli and shown to efficiently degrade chlorite with an activity optimum at pH 5 (kcat 1144 ± 23.8 s-1, KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106 M-1 s-1). The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7. Cyanide mediates the formation of a low-spin complex with kon = (1.6 ± 0.1) × 105 M-1 s-1 and koff = 1.4 ± 2.9 s-1 (KD ~ 8.6 μM). Both, thermal and chemical unfolding follows a non-two state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2-producing proteins in (nitrogen-fixing) cyanobacteria

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high resolution crystal structures of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 \uc5) and the variants D143A (1.3 \uc5), R232A (1.9 \uc5), and D143A/R232A (1.1 \uc5). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp 143 and Arg 232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin (LS) cyanide complex as well as on (iv) the stability and reactivity of an oxoiron(IV)porphyrin \u3c0-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527 and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs

Molecular mechanism of enzymatic chlorite detoxification: insights from structural and kinetic studies

The heme enzyme chlorite dismutase (Cld) degrades chlorite to chloride and dioxygen. Although the structure and steady-state kinetics of pentameric Clds have been elucidated, many questions remain, such as the mechanism of chlorite cleavage and the pH dependence of the reaction. Here, we present high resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple and the UV-vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite cannot recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during reaction. Homolytic cleavage of chlorite is propose

Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry and Reactivity

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from \u201cCandidatus Nitrospira defluvii\u201d (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV\u2013vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O\u2550O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E\ub0\u2032 = 12113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation