Publications

Interaction studies of biliverdin-Cu complex formation using XANES, XRF, and DFT analyses and its effect on cytotoxicity in glioblastoma cell line (U251)

Cu2+ and biliverdin build complex (BV–Cu) that is stable in water at physiological pH and may develop in living systems. However, the interactions of BV-Cu with cells and even its structure are not fully understood. Herein we showed that BV-Cu complex was significantly more cytotoxic to a human cell line than equimolar Cu2+ and biliverdin. Micro-XRF-generated maps of elements showed that in contrast to Cu2+-treated cells which exhibited physiological compartmentalization of Cu, cells treated with BV-Cu had almost even distribution of Cu throughout the cytosol. XANES showed that the complex contains Cu1+ with distorted tetrahedral or square planar geometry. This implicates that the formation of the complex includes redox reaction between the metal ion and the ligand. As a result, the complex is nominally made of Cu1+ and BV radical cation. The redistribution of charge and spin densities between Cu2+ and tetrapyrrole ring during complex formation was also corroborated by DFT modelling. Cells exposed to BV-Cu were loaded with Cu that remained in 1+ form with altered local geometry than the complex. The cytotoxicity of the BV-Cu complex may come as a result of its interference with regular Cu transport and storage routes.

Employing microalga Chlorella sorokiniana in the biosynthesis of paramagnetic and catalytically functional manganese cluster

Finding vehicles for biosynthesis of metal clusters with advantageous magnetic and catalytic properties is an important industrial and environmental task. We have found previously that green microalga Chlorella sorokiniana produces a multivalent Mn-O cluster with structure that is similar to photosynthetic oxygen-evolving complex (OEC). Here we reported magnetic and redox properties and the site of accumulation of this cluster, and we proposed the mechanisms of biosynthesis and the protocol for extraction. The cluster was paramagnetic even at room temperature, with an antiferromagnetic transition at ∼13 K. The separation between ground and excited state of ΔE ≈ 15.0 cm−1 matched the separation energy of OEC in S2 state. Nano X-ray fluorescence microscopy and 31P NMR showed that the cluster is accumulated in acidocalcisomes, a lysosome-type organelles rich in polyphosphates. The conditions in these organelles resemble the settings of chemical synthesis of OEC mimics, including mildly acidic pH and the availability of Ca2+ ions. Polyphosphates are likely to play a role of stabilizing ligands and modulators of redox properties of Mn2+ in the cluster synthesis. The cluster shares redox potentials with OEC and showed catalase-like activity. However, we could not confirm OEC-like performance because the cluster was prone to degradation by oxidizing agents in the presence of organic residue in the extract. The biosynthesis showed an overall yield of ∼25 % and appears to be cost-competitive with chemical synthesis. This study shows that metabolic trades of selected microalgae can be employed in the green synthesis of catalytically functional clusters.

The [2Fe‐2S] cluster of mitochondrial outer membrane protein mitoNEET has an O2‐regulated nitric oxide access tunnel

The mitochondrial outer membrane iron–sulphur ([Fe‐S]) protein mitoNEET has been extensively studied as a target of the anti‐inflammatory and type‐2 diabetes drug pioglitazone and as a protein affecting mitochondrial respiratory rate. Despite these extensive past studies, its molecular function has yet to be discovered. Here, we applied an interdisciplinary approach and discovered an explicit nitric oxide (NO) access site to the mitoNEET [2Fe‐2S] cluster. We found that O2 and pioglitazone block NO access to the cluster, suggesting a molecular function for the mitoNEET [2Fe‐2S] cluster in mitochondrial signal transduction. Our discovery hints at a new pathway via which mitochondria can sense hypoxia through O2 protection of the mitoNEET [2Fe‐2S] cluster, a new paradigm in understanding the importance of [Fe‐S] clusters for gasotransmitter signal transduction in eukaryotes.

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at −31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2

Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron–sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron–sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein–protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.

Shedding Light on the Electron Delocalization Pathway at the [Fe2S2]2+ Cluster of FDX2

In this paper, we investigate the electronic structure of the [Fe2S2]2+ cluster of human ferredoxin 2 by designing NMR experiments tailored to observe hyperfine-shifted and fast relaxing resonances in the immediate proximity of the cluster and adding a quantitative layer of interpretation through quantum chemical calculations. The combination of paramagnetic NMR and density functional theory data provides evidence of the way unpaired electron density map is at the origin of the inequivalence of the two iron(III) ferredoxin centers. An electron spin density transfer is observed between cluster inorganic sulfide ions and aliphatic carbon atoms, occurring via a C–H---S–Fe3+ interaction, suggesting that inorganic cluster sulfide ions have a significant role in the distribution of electron spin density around the prosthetic group. The extended assignment of 1H, 13C, and 15N nuclei allows the identification of all residues of the binding loop and provides an estimate of the magnetic exchange coupling constant between the two Fe3+ ions of the [Fe2S2]2+ cluster of 386 cm–1. The approach developed here can be extended to other iron–sulfur proteins, providing a crucial tool to uncover subtle differences in electronic structures that modulate the functions of this protein family.

Optimized 13C Relaxation-Filtered Nuclear Magnetic Resonance: Harnessing Optimal Control Pulses and Ultra-High Magnetic Fields for Metalloprotein Structural Elucidation

Ultra-high magnetic fields and high-sensitivity cryoprobes permit the achievement of a high S/N ratio in 13C detection experiments, thus making a 13C superWEFT (Super water eliminated Fourier transform) experiment feasible. 13C signals that are not visible using 1H observed heteronuclear experiments, nor with established 2D 13C direct detection experiments, become easily observable when a 13C relaxation-based filter is used. Within this frame, optimal control pulses (OC pulses) have been, for the first time, applied to paramagnetic systems. Although the duration of OC pulses competes with relaxation, their application to paramagnetic signals has been successfully tested. OC pulses are much more efficient with respect to the phase- and amplitude-modulated ones routinely used at lower fields while providing bandwidth excitation profiles that are sufficient to meet the need to cover up to an 80 ppm spectral region. On the other hand, when paramagnetic relaxation is shorter than the duration of OC pulses, the use of hard, rectangular pulses is, at the present state of the art, the best approach to minimize the loss of signal intensity.