Metal-catalyzed Oxidation of Histidine in Human Growth Hormone

Abstract

In physiological compartments, provided with suitable reducing agents, transition metals are able to catalyze the activation of O 2 to various reactive oxygen species (ROS) 1 which readily attack biomolecules. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress and aging where modified proteins may accumulate in tissue (1-9). In addition, MCO also presents a stability problem for the biotechnological manufacturing of proteins (10, 11).A unique feature often observed during MCO of proteins is that only a few amino acid residues within a certain domain of the given protein are modified (2, 12). Such site specificity is attributed to the generation of ROS at specific metal-binding sites, where the highly reactive ROS attack labile functional groups nearby rather than diffuse into the bulk medium. It has been shown that most of the enzymes labile to MCO require metals for activity (13). The oxidation of these enzymes is less sensitive to the inhibition by exogenous ROS scavengers than would be expected on the basis of known rate constants for the reactions of specific ROS with these scavengers. However, one problem associated with all these studies is that neither the oxidizing species nor the intermediary protein-transition metal complexes formed during MCO of proteins have been well characterized. This is most pertinent to copper-catalyzed oxidation (i.e. to the frequently used ascorbate/Cu(II)/O 2 system) involving the Cu(II)/Cu(I) redox couple (2). Here, Cu(II) will most probably require a different geometry and different ligands within the protein ligand sphere than Cu(I). An initial reduction of Cu(II), bound to a metal-binding site, to Cu(I) may change the geometry of the protein-metal complex, and possibly even release Cu(I) from the metal-binding site. On the other hand, the metal may be retained when (i) the protein is flexible enough to adapt to the new geometrical requirements of the reduced metal, or (ii) re-oxidation of the metal by molecular oxygen or reduction products of molecular oxygen such as superoxide or hydrogen peroxide occurs faster than the release of the reduced metal from the metal-binding site.In a recent example for electron transfer coupled ligand dynamics in the model complex Cu(I/II)(TTCN) 2 , we could show that oxidation of tetrahedral [Cu(I)(TTCN) 2 ] ϩ potentially led to the loss of a ligand to yield [Cu(II)(TTCN)(H 2 O) 3 ] 2ϩ before geometrical reorganization around the Cu(II) atom allowed the addition of TTCN to give the final product, octahedral [Cu(II)(TTCN) 2 ] 2ϩ . 2 Analogous mechanisms may lead to kinetic complexities during protein oxidation which may be reflected in oxidation yields and kinetics as well as the nature of the oxidation products.One amino acid particularly affected by MCO is histidine (His) (15-17). There is as yet no detailed mechanism available for His oxidation in proteins, except a crystallographic study which showed the feasibility of metal-catalyzed processes within the metal-binding domain of glutamine syn...

Extracellular Superoxide Dismutase Inhibits Inflammation by Preventing Oxidative Fragmentation of Hyaluronan

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“…Representative UV traces of peptide maps of native, NaOCl-and MPO-modified lipid-free apo A-I are shown in Figure 4. To identify tryptic fragments of native apo A-I, RP-HPLC separation was followed by online ESI-MS analyses in a first set of experiments, an analytical tool successfully applied to the identification of tryptic peptides obtained from digests of different proteins [35]. The residue number, sequence and observed molecular masses for the corresponding tryptic peptides of native apo A-I ( Figure 4A) are summarized in Table 2.…”

Mass spectrometry in aging research

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