Human endonuclease III/NTH1: focusing on the [4Fe-4S] cluster and the N-terminal domain.

Human Endonuclease III (EndoIII), hNTH1, is an FeS containing enzyme which repairs oxidation damaged bases in DNA. We report here the first comparative biophysical study of full-length and an N-terminally truncated hNTH1, with a domain architecture homologous to bacterial EndoIII. Vibrational spectroscopy, spectroelectrochemistry and SAXS experiments reveal distinct properties of the two enzyme forms, and indicate that the N-terminal domain is important for DNA binding at the onset of damage recognition.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies.

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN- ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.

Protonation of the Cysteine Axial Ligand Investigated in His/Cys c-Type Cytochrome by UV-Vis and Mid- and Far-IR Spectroscopy.

His/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Toward a better understanding of Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and mid- to far-FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C = -375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys ν(SH), His ν(C5N1), heme δ(CmH)) and far-IR (ν(Fe(II)-His), ν(His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, is stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination.

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties.

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = -440 mV, is lower than that of Fd1, Em = -372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Šresolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.

Gold and Silver Nanoparticles for Applications in Theranostics.

Noble metal nanomaterials, such as gold or silver nanoparticles, exhibit unique photonic, electronic, catalytic and therapeutic properties. The high versatility in their synthesis, especially size and shape features, as well as in the surface functionalization by, e.g., physisorption, direct chemisorption of thiol derivatives and covalent binding through bifunctional linkers or specific affinity interactions, prompted their widespread and rising use as multifunctional platforms for theranostic purposes. In this paper, the recent developments of gold and silver nanoparticles for application in biosensing, medical imaging, diagnosis and therapy is reviewed from the following five aspects: (1) the gold and silver nanomaterials intrinsic properties of biomedical interest; (2) the synthesis of noble metal nanoparticles by chemical, physical and biological/green processes; (3) the applications of gold and silver nanoparticles in imaging, diagnostic and therapeutic mode; (4) the surface functionalization processes for targeting, controlled drug loading and release, triggered pathways of cellular uptake and tissue distribution; and (5) nanotoxicity. The historical developments and the most recent applications have been focused on, together with suggested strategies for future more efficacious, targeted delivery.