Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat =3.9(4) s-1) lower than that of wt SmNir (kcat =240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H...OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H...OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.Fil: Cristaldi, Julio César. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Gómez, María C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Ferroni, Felix Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Dalosto, Sergio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; ArgentinaFil: Rizzi, Alberto Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Brondino, Carlos Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentin

Implications of Metal Coordination in Damage and Recognition of Nucleic Acids and Lipid Bilayers

Metal ions have a myriad of biological functions from structural stability to enzymatic (de)activation and metabolic electron transfer. Redox-active metals also mediate the formation of reactive oxygen species which may either cause oxidative damage or protect cellular components. Computational modeling is used here to investigate the role of (1) metal-ion binding to antimicrobial peptides, (2) metal-ion removal and disulfide formation on zinc finger (ZF) proteins, and (3) coordination of thiones/selones for the prevention of metal-mediated redox damage. Piscidins, natural-occurring antimicrobial peptides, efficiently kill bacteria by targeting their membranes. Their efficacy is enhanced in vitro by metal-binding and the presence of membrane-destabilizing oxidized phospholipids (oxPLs). Molecular dynamics (MD) simulations are used to model insertion of Ni2+-bound piscidins 1 (P1:Ni2+) and 3 (P3:Ni2+) into lipid bilayers in the presence and absence of oxPLs. Metallation promotes deeper peptide insertion in the membrane, and P1:Ni2+ is suggested to interact more with anionic lipid headgroups in the presence of oxPLs. The release of Zn2+ from ZF proteins through oxidation of the cysteine thiolates is associated with inhibition of viral replication, disruption of cancer gene expression, and DNA repair preventing tumor growth. Multi-microsecond MD simulations were performed to examine the effect of cysteine oxidation on the ZF456 fragment of transcription factor IIIA and its complex with 5S RNA. Upon oxidation in the absence of RNA, the individual ZF domains unfold yielding a globular ZF456 peptide. Oxidation of the RNA-bound ZF456 peptide disrupts key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. The antioxidant properties of sulfur and selenium compounds prevent metal-mediated (i.e., Fenton chemistry) oxidative damage. The effect of the coordination of sulfur/selenium derivatives of imidazolidinone (thiones/selones) on the electronic structure and reduction potential of Fe2+ ions solvated or coordinated to guanine are examined using density functional theory. The highest occupied molecular orbital (HOMO) for the Fe(II)-aqua complex is metal-centered but localized on the nucleobase in the Fe2+-guanine complex. Complexation of the thione/selone shifts the HOMO to the sulfur/selenium center suggesting a mechanism for protection of DNA by sacrificial oxidation of the sulfur/selenium ligand

Benchmarking of copper(II) LFMM parameters for studying amyloid-β peptides

Ligand field molecular mechanics (LFMM) parameters have been benchmarked for copper (II) bound to the amyloid-β1–16 peptide fragment. Several density functional theory (DFT) optimised small test models, representative of different possible copper coordination modes, have been used to test the accuracy of the LFMM copper bond lengths and angles, resulting in errors typically less than 0.1 Å and 5°. Ligand field molecular dynamics (LFMD) simulations have been carried out on the copper bound amyloid-β1–16 peptide and snapshots extracted from the subsequent trajectory. Snapshots have been optimised using DFT and the semi-empirical PM7 method resulting in good agreement against the LFMM calculated geometry. Analysis of substructures within snapshots shows that the larger contribution of geometrical difference, as measured by RMSD, lies within the peptide backbone, arising from differences in DFT and AMBER, and the copper coordination sphere is reproduced well by LFMM. PM7 performs excellently against LFMM with an average RMSD of 0.2 Å over 21 tested snapshots. Further analysis of the LFMD trajectory shows that copper bond lengths and angles have only small deviations from average values, with the exception of a carbonyl moiety from the N-terminus, which can act as a weakly bound fifth ligand

Computer-aided laccase engineering: toward biological oxidation of arylamines

Oxidation of arylamines, such as aniline, is of high industrial interest and laccases have been proposed as biocata-lysts to replace harsh chemical oxidants. However, the reaction is hampered by the redox potential of the substrate at acid pH and enzyme engineering is required to improve the oxidation. In this work, instead of trying to improve the redox potential of the en-zyme, we aim towards the (transient) substrate’s one and propose this as a more reliable strategy. We have successfully combined a computational approach with experimental validation to rationally design an improved biocatalyst. The in silico protocol combines classical and quantum mechanics to deliver atomic and electronic level detail on the two main processes involved: substrate binding and electron transfer. After mutant expression and comparison to the parent type, kinetic results show that the protocol accurately predicts aniline’s improved oxidation (2-fold kcat increase) in the engineered variant for biocatalyzed polyaniline production.This study was supported by the INDOX (KBBE-2013-7-613549) EU-project, and the NOESIS (BI0201456388-R) and OxiDesign (CTQ2013-48287-R) Spanish project. GS thanks an FPI grant of the Spanish Ministry of Competitiveness.Peer ReviewedPostprint (author's final draft

Computational study of the structure and electronic circular dichroism spectroscopy of blue copper proteins

The calculation of the electronic circular dichroism (CD) spectra of the oxidised form of the blue copper proteins plastocyanin and cucumber basic protein and the relationship between the observed spectral features and the structure of the active site of the protein is investigated. Excitation energies and transition strengths are computed using multi reference configuration interaction, and it is shown that computed spectra based on coordinates from the crystal structure or a single structure optimised in quantum mechanics/molecular mechanics (QM/MM) or ligand field molecular mechanics (LFMM) are qualitatively incorrect. In particular, the rotational strength of the ligand to metal charge transfer band is predicted to be too small or have the incorrect sign. By considering calculations on active site models with modified structures it is shown that the intensity of this band is sensitive to the non-planarity of the histidine and cysteine ligands coordinated to copper. Calculation of the ultraviolet absorption and CD spectra based upon averaging over many structures drawn from a LFMM molecular dynamics simulation are in good agreement with experiment, and superior to analogous calculations based upon structures from a classical molecular dynamics simulation. This provides evidence that the LFMM force field provides an accurate description of the molecular dynamics of these proteins

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry

Structure of the Intermolecular Complex between Plastocyanin and Cytochrome f from Spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin