Dömötör, O.; Enyedy, É.A.

J. Biol. Inorg. Chem. 2019, 24, 703-719, 10.1007/s00775-019-01683-0

Various half-sandwich ruthenium(II) arene complexes and rhodium(III) arene complexes have been intensively investigated due to their prominent anticancer activity. The interaction of the organometallic complexes of Ru(η6-p-cymene) and Rh(η5-C5Me5) with human serum albumin (HSA) was studied in detail by a combination of various methods such as ultrafiltration, capillary electrophoresis, 1H NMR spectroscopy, fluorometry and UV–visible spectrophotometry in the presence of 100 mM chloride ions. Binding characteristics of the organometallic ions and their complexes with deferiprone, 2-picolinic acid, maltol, 6-methyl-2-picolinic acid and 2-quinaldic acid were evaluated. Kinetic aspects and reversibility of the albumin binding are also discussed. The effect of low-molecular-mass blood components on the protein binding was studied in addition to the interaction of organorhodium complexes with cell culture medium components. The organometallic ions were found to bind to HSA to a high extent via a coordination bond. Release of the bound metal ions was kinetically hindered and could not be induced by the denaturation of the protein. Binding of the Ru(η6-p-cymene) triaqua cation was much slower (ca. 24 h) compared to the rhodium congener (few min), while their complexes interacted with the protein relatively fast (1–2 h). The studied complexes were bound to HSA coordinatively. The highly stable and kinetically inert 2-picolinate Ru(η6-p-cymene) complex bound in an associative manner preserving its original entity, while lower stability complexes decomposed partly or completely upon binding to HSA. Fast, non-specific and high-affinity binding of the complexes on HSA highlights their coordinative interaction with various types of proteins possibly decreasing effective drug concentration.

Emerson, J.P.

J. Biol. Inorg. Chem. 2013, 18, 595-598, 10.1007/s00775-013-1009-1

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu2+ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (K a approximately 5 × 1012 and 1 × 1010), and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

Hirota, S.; Lin, Y.-W.

J. Biol. Inorg. Chem. 2018, 23, 7-25, 10.1007/s00775-017-1506-8

Noncovalent weak interactions [hydrophobic interaction and hydrogen (H)-bond] play crucial roles in controlling the functions of biomolecules, and thus have been used to design artificial metalloproteins/metalloenzymes during the past few decades. In this review, we focus on the recent progresses in protein design by tuning the noncovalent interactions, including hydrophobic and H-bonding interactions. The topics include redesign and reuse of the heme pocket and other protein scaffolds, design of the heme protein interface, and de novo design of metalloproteins. The informations not only give insights into the metalloenzyme reaction mechanisms but also provide new reactions for future applications.

Imperiali, B.

Prot. Eng. 1997, 10, 691-698, 10.1093/protein/10.6.691

The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam'. The chimeric Pam residue integrates the heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position. The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme structure/ function relationships in a native-like peptidyl architecture.