The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

We report here two copper complexes as first functional models for lytic polysaccharide monooxygenases, mononuclear copper-containing enzymes involved in recalcitrant polysaccharide breakdown. These complexes feature structural and spectroscopic properties similar to those of the enzyme. In addition, they catalyze oxidative cleavage of the model substrate p-nitrophenyl-β-d-glucopyranoside. More importantly, a particularly stable copper(II) hydroperoxide intermediate is detected in the reaction conditions.

Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

DAPI (4',6-diamidino-2-phenylindole) is a widely used fluorescent dye, whose complicated binding features to DNAs and RNAs have been the object of debates and are still not fully understood. In this study, different approaches were employed, including binding equilibrium measurements (spectrofluorometry), melting experiments (spectrophotometry), viscometric measurements, circular dichroism, and T-jump kinetic analyses; all data concur in shedding light on the complex mechanistic aspects of the binding mode of DAPI to natural DNA. Conditions are found that induce the mode of the DAPI/DNA interaction to change from groove binding to intercalation. Moreover, it is observed, for the first time, that DAPI is able to induce the formation of a rather compact polymer-dye adduct under particular conditions. The results suggest that this form is a folded or coiled DNA structure stabilized by DAPI dye bridges.

Identification of stable HOPO–UO22+–fetuin ternary complexes after a chromatographic separation process.

Uranium speciation and bioaccumulation were investigated in the sea urchin Paracentrotus lividus. Through accumulation experiments in a well-controlled aquarium followed by ICP-OES analysis, the quantification of uranium in the different compartments of the sea urchin was performed. Uranium is mainly distributed in the test (skeletal components), as it is the major constituent of the sea urchin, but in terms of quantity of uranium per gram of compartment, the following rating: intestinal tract > gonads >> test, was obtained. Combining both extended X-ray Absorption Spectroscopy (XAS) and time resolved laser induced fluorescence (TRLFS) spectroscopic analysis, it was possible to identify two different forms of uranium in the sea urchin, one in the test, as a carbonato-calcium complex, and the second one in the gonads and intestinal tract, as a protein complex. Toposome is a major calcium-binding transferrin-like protein contained within the sea urchin. EXAFS data fitting of both contaminated organs in vivo and the uraniumtoposome complex from protein purified out of the gonads revealed that it is suspected to complex uranium

The equilibria and kinetics (stopped-flow) of the binding of Ni(II) to salicylhydroxamic acid (SHA) and phenylbenzohydroxamic acid (PBHA) have been investigated in aqueous solutions containing SDS micelles. The two ligands are fairly distributed between the two pseudophases present, so the binding reaction occurs in both phases. The contributions to the total reaction from each phase has been evaluated, following a procedure where use is made of the experimentally determined partition coefficients of the reactants involved. The mechanism of the reaction occurring on the micelle surface has been derived and comparison with the mechanism in water shows that the step Ni(2+) + HL ⇄ NiHL(2+) is operative in both pseudophases, whereas the step Ni(2+) + L(-)⇄ NiL(+), which is operative in water, is replaced in SDS by the step NiOH(+) + HL ⇄ NiL(+). The analysis of the equilibrium and of the kinetic data enabled the evaluation of the equilibrium and the rate constants of the individual steps taking part in the binding process over the micelle surface. Interestingly, the first hydrolysis constant of the Ni(H(2)O)(6)(2+) ion in SDS is more than two orders of magnitude higher than in water. The agreement between the equilibrium constants derived from kinetics and those obtained by static measurements confirms the validity of the proposed mechanism.

The equilibria and the kinetics of the binding of Iron(III) to salicylhydroxamic (SHA) and benzohydroxamic (BHA) acids have been investigated in aqueous solution (I = 1 M (HClO(4)/NaClO(4)), T = 298 K) using spectrophotometric and stopped-flow methods. Whereas Iron(III) forms a 1:1 complex (ML) with BHA, it forms both ML and M(2)L complexes with SHA. The presence of M(2)L in aqueous medium is corroborated by FTIR measurements. The reactive form of Iron(III) is the hydrolyzed species FeOH(2+), which binds to the O,O site in ML and to the O,O and O(P),N (P = phenolate) sites in M(2)L, inducing full deprotonation of the latter. The reaction pathway is discussed in terms of a multistep mechanistic scheme in which the metal-ligand interaction is coupled to hydrolysis and self-aggregation steps of Iron(III). The observation and characterization of M(2)L as a stable species is important because it contains the -Fe-O-N-Fe- sequence, which constitutes the repetitive motif of the SHA-based metallacrown ring and provides the rationale for 12-MC-4 metallacrowns. In the framework of this study, the kinetics of the Iron(III) dimerization and trimerization have also been investigated using the stopped-flow method to perform dilution jumps. The reaction scheme put forward involves two parallel steps (FeOH(2+) + FeOH(2+) and Fe(3+) + FeOH(2+)) that lead to formation of the Fe(2)(OH)(2)(4+) dimer and a slower step (FeOH(2+) + Fe(2)(OH)(2)(4+)) to form the trimer species. The kinetics of the last step have been investigated here for the first time, and the results deduced indicate that, of the two possible trimer structures reported in the literature, Fe(3)(OH)(3)(6+) and Fe(3)(OH)(4)(5+), the latter prevails by far.