Radu Silaghi-Dumitrescu Laboratory

Research in our lab is mainly focused on small molecule activation at bioinorganic systems containing transition metals (heme proteins, non-heme iron cobalamin, copper-containing proteins, model systems and others) as well as on oxidative/nitrosative stress especially when pertaining to bioinorganic centers and biologically-relevant free radical chemistry.

We typically employ methods such as UV-vis, EPR, stopped-flow, freeze-quench, molecular biology, protein overexpression and purification, as well as molecular modelling (DFT, semiempirical, molecular dynamics, docking and others).

Current research projects:

The blood currently used in transfusions presents problems linked to stability (requiring refrigeration and becoming outdated after about a month), availability (especially under crisis situations, such as major accidents, military situations, or major outbreaks), antigens (blood group compatibility), contamination (AIDS, hepatitis, or agents yet to be identified) or ethics (e.g., Jehovah's witnesses). Blood substitutes are chemical or biochemical preparations aimed at being used in transfusions without the above-mentioned drawbacks and with the sole role of enhancing oxygen transport by the patient's blood (as opposed to the more complex functions that blood normally achieves, such as transport or defense).

There are three classes of blood substitutes currently being debated in the scientific literature. The first two classes represent purely synthetic approaches, while the third is semi synthetic, employing the hemoglobin already produced by living organisms. We have recently added a fourth class of blood substitutes, whose active ingredient is also a protein specialized in oxygen transport, but of non-heme nature: hemerythrin (Hr). Hr employs a binuclear non-heme diferrous center to bind oxygen; the resulting adduct is formally described as Fe(III)-Fe(III)-OOH, i.e. diferric peroxo. This mode of binding oxygen has the remarkable advantage of (1) not featuring a superoxide ligand (unlike Hb), which drastically reduces the reactivity towards NO, and (2) exhibiting stability towards peroxide (unlike Hb).

Cisplatin and related compounds are known to exert much of their useful therapeutic effects via binding to DNA. The need for more effective drugs as well as the wide range of side-effects (nausea, progressive peripheral sensory neuropathy, fatigue, vomiting, alopecia, hematological suppression, renal damage) have, for several decades now, fuelled interest into understanding the complex mechanisms of interaction of cisplatin and related compounds with various biomolecules. One notable observation in this respect has been that platinum can bind to a range of proteins, as demonstrated by elemental analyses, chromatography and mass spectrometry. Our own research, with established as well as with experimental drugs, including natural extracts of various sources, is seeking to investigate the free-radical mechanisms involved in such drug-protein interactions, with purified proteins as well as with intact cells.

Reduction of nitrite to nitric oxide is essential in certain living species, and has recently been proposed to be an important secondary function of hemoglobin in humans. In vivo, nitrite reduction is accomplished by metalloenzymes, involving direct metal-nitrite coordination. We proposed that linkage nitro/nitrito isomerism is an essential part of the mechanism in copper and in heme d1-containing nitrite reductases. Recent experimental work has also shown involvement of linkage isomerism in catalytic reduction of nitrite by free hemes and related small complexes in solution. This mechanism reconciles the cd1NIR chemistry with the puzzling fact that Fe(III)-NO is kinetically inert and hence cannot possibly be a part of the cd1NIR catalytic cycle. cycle

Proteins such as cytochrome P450 (P450), horseradish peroxidase (HRP), hemoglobin and catalase are all known to bind and/or ruduce (activate) dioxygen at their heme active sites, in the ferrous form. A brief overview of these process is given below.

reactions of active site with O<sub>2</sub> and/or O<sub>2</sub>H<sub>2</sub>. “S” denotes an organic
substrate molecule. “X” may be a protein-derived cysteinate or histidine

Our interest has been in defining the properties and reactivity of the transient adducts depicted in the Figure above (nature of ferrous-dioxygen bonding, nature of ferric-peroxo interaction, electronic structure and protonation state of ferryl, i.e. Compounds I and II).

We are also pursuing the related reactions of heme systems with sulfide and sulfur oxyanions (e.g., sulfite reductase and sulfhemoglobin chemistry), as well as with halide oxyanions (relevant to myeloperoxidase, chlorite discutase and others). We have more recently extended this line towards other transition metal - containing macrocyclic complexes, such as cobalamin, porphyrazines and phthalocyanines.
In such systems, unusual metal oxidation states (e.g., Fe(IV), Fe(I), Fe(0), Co(I)) under physiologically-relevant conditions are a common theme.

Laccase (p-diphenol:dioxygen oxidoreductase), one of the oldest discovered enzymes, contains four copper ions in two active sites and catalyzes a monoelectronic oxidation of substrates such as phenols and their derivatives, or aromatic amines, coupled to a fou relectron reduction of dioxygen to water. The catalytic mechanism was studied for decades but is still not completely elucidated, especially in terms of the reduction of dioxygen to water. The high interest in laccases is explained by the large number of biotechnological applications. We have purified and characterized laccase-type enzymes, with interest in their applications as well as in further understanding their mechanisms of action.

Crystal structure of the AmtB trimer

Polyoxometalates (POMs) comprise a rich and diverse family of metal-oxygen clusters of the early transition metals in high oxidation states, most commonly V(V), Mo(VI) and W(VI), with unique versatility in terms of shape, polarity, redox potentials, surface charge distribution, acidity, and solubility. We are analyzing iron-substituted POMs in search of features common with/reminiscent of those seen in iron-based redox metalloenzymes.

We are investigating preferences for various types of secondary structure, and tools for predicting three-dimensional structures in peptides and in other biopolymers, such as polilactic acid and polymers relevant in dental materials.

We are examining plant extracts, fungi, food- or drug-related components, or other natural products, for their antioxidant and biological activity (anticancer, etc.), with emphasis on chemical mechanisms of their action.

We are also pursuing the application of chemical and biochemical concepts in social/cultural studies.

© Radu Silaghi-Dumitrescu 2019