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Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

ABSTRACT: The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh₂(?-O₂CCH₃)₄] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh₂(?-O₂CCH₃)₄] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side chains of Arg14 and His15. Dimeric Rh-Rh units with Rh-Rh distances between 2.3 and 2.5 Å are bound to the side chains of Asp18, Asp101, Asn93, and Lys96, while a dirhodium unit with a Rh-Rh distance of 3.2-3.4 Å binds the C-terminal carboxylate and the side chain of Lys13 at the interface between two symmetry-related molecules. An additional monometallic fragment binds the side chain of Lys33. These data, which are supported by replicated structural determinations, shed light on the reactivity of dirhodium tetracarboxylates with proteins, providing useful information for the design of new Rh-containing biomaterials with an array of potential applications in the field of catalysis or of medicinal chemistry and valuable insight into the mechanism of action of these potential anticancer agents.

SUBMITTER: Loreto D

PROVIDER: S-EPMC7867343 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

Loreto Domenico D Ferraro Giarita G Merlino Antonello A

International journal of molecular sciences 20210202 3

The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh2(μ-O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh2(μ-O2CCH3)4] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side c ...[more]

PMID: 33540880

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Project description:We have combined results from several spectroscopic techniques to investigate the aerobic reactions of Rh2(AcO)4 (AcO- = CH3COO-) with l-cysteine (H2Cys) and its derivatives d-penicillamine (3,3'-dimethylcysteine, H2Pen), with steric hindrance at the thiol group, and N-acetyl-l-cysteine (H2NAC), with its amino group blocked. Previous investigations have shown that antitumor active dirhodium(II) carboxylates may irreversibly inhibit enzymes containing a thiol group at or near their active sites. Also, cysteine, the only thiol-containing proteinogenic amino acid, interacts in vivo with this class of antitumor compounds, but structural information on the products of such reactions is lacking. In the present study, the reactions of Rh2(AcO)4 and H2L were carried out in aqueous solutions at the pH of mixing (acidic) and at physiological pH, using the different mole ratios 1:2, 1:4, and 1:6, which resulted in the same products in increasing yields. Electrospray ionization mass spectrometry (ESI-MS) indicates formation of dimeric [RhIII 2Pen4]2- or oligomeric {RhIII 2L4} n (L = Cys, NAC) complexes with bridging thiolate groups. Analyses of Rh K edge extended X-ray absorption fine structure (EXAFS) data reveal 3-4 Rh-S and 2-3 Rh-(N/O) bonds around six-coordinated Rh(III) ions at mean distances of 2.33 ± 0.02 and 2.09 ± 0.02 Å, respectively. In the N-acetyl-l-cysteine compound, the RhIII···RhIII distance 3.10 ± 0.02 Å obtained from the EXAFS spectrum supports trithiolate bridges between the Rh(III) ions, as was also found when using glutathione as ligand. In the cysteine and penicillamine complexes, double thiolate bridges join the Rh(III) ions, with the nonbridging Cys2- and Pen2- ligands in tridentate chelating (S,N,O) mode, which is consistent with the ΔδC = 7.3-8.4 ppm shift of the COO- signal in their carbon-13 cross polarization magic angle spinning (CPMAS) NMR spectra. For the penicillamine complex, the 2475.6 eV peak in its S K edge X-ray absorption near edge structure (XANES) spectrum shows partial oxidation, probably caused by peroxide generated from reduction of dissolved O2, of thiolato to sulfenato (S=O) groups, which were also identified by ESI-MS for all three {RhIII 2L4} n compounds.

Project description:In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

Dataset Information

Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

Publications

Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

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