Dopamine (DA) is the most important catecholamine in the brain, as it is the most abundant and the precursor of other neurotransmitters. Degeneration of nigrostriatal neurons of substantia nigra pars compacta in Parkinson's disease represents the best‐studied link between DA neurotransmission and neuropathology. Catecholamines are reactive molecules that are handled through complex control and transport systems. Under normal conditions, small amounts of cytosolic DA are converted to neuromelanin in a stepwise process involving melanization of peptides and proteins. However, excessive cytosolic or extraneuronal DA can give rise to nonselective protein modifications. These reactions involve DA oxidation to quinone species and depend on the presence of redox‐active transition metal ions such as iron and copper. Other oxidized DA metabolites likely participate in post‐translational protein modification. Thus, protein–quinone modification is a heterogeneous process involving multiple DA‐derived residues that produce structural and conformational changes of proteins and can lead to aggregation and inactivation of the modified proteins.

Metmyoglobin catalyzes the nitration of various phenolic compounds in the presence of nitrite and hydrogen peroxide. The reaction rate depends on the reactant concentrations and shows saturation behavior. Two competing paths are responsible for the reaction. In the first, myoglobin reacts according to a peroxidase-like cycle forming two active intermediates, which can induce one-electron oxidation of the substrates. The MbFe(IV)==O intermediate oxidizes nitrite to nitrogen dioxide, which, after reaction with the phenol or with a phenoxy radical, yields the nitrophenol. In the second mechanism, hydrogen peroxide reacts with iron-bound nitrite to produce an active nitrating species, which we assume to be a protein-bound peroxynitrite species, MbFe(III)--N(O)OO. The high nitrating power of the active species is shown by the fact that the catalytic rate constant is essentially independent of the redox properties of the phenol. The occurrence of one or other of these mechanisms depends on the nitrite concentration: at low [NO(2) (-)] the nitrating agent is nitrogen dioxide, whereas at high [NO(2) (-)] the peroxynitrite path is dominant. The myoglobin derivative that accumulates during turnover depends on the mechanism. When the path involving NO(2) (.) is dominant, the spectrum of the MbFe(IV)==O intermediate is observed. At high nitrite concentration, the Soret band appears at 416 nm, which we attribute to an iron-peroxynitrite species. The metMb/NO(2) (-)/H(2)O(2) system competitively nitrates the heme and the endogenous tyrosine at position 146 of the protein. Phenolic substrates protect Tyr146 from nitration by scavenging the active nitrating species. The exposed Tyr103 residue is not nitrated under the same conditions.

The modification of human myoglobin (HMb) by reaction with nitrite and hydrogen peroxide has been investigated. This reaction is important because NO(2) (-) and H(2)O(2) are formed in vivo under conditions of oxidative and nitrative stress, where protein derivatization has been often observed. The abundance of HMb in tissues and in the heart makes it a potential source and target of reactive species generated in the body. The oxidant and nitrating species produced by HMb/H(2)O(2)/NO(2) (-) are nitrogen dioxide and peroxynitrite, which can react with exogenous substrates and endogenous protein residues. Tandem mass analysis of HMb modified by stoichiometric amounts of H(2)O(2) and NO(2) (-) indicated the presence of two endogenous derivatizations: oxidation of C110 to sulfinic acid (76 %) and nitration of Y103 to 3-nitrotyrosine (44 %). When higher concentrations of NO(2) (-) and H(2)O(2) were used, nitration of Y146 and of the heme were also observed. The two-dimensional gel-electrophoretic analysis of the modified HMbs showed spots more acidic than that of wild-type HMb, a result in agreement with the formation of sulfinic acid and nitrotyrosine residues. By contrast, the reaction showed no evidence for the formation of protein homodimers, as observed in the reaction of HMb with H(2)O(2) alone. Both HMb and the modified HMb are active in the H(2)O(2)/NO(2) (-)-dependent nitration of exogenous phenols. Their catalytic activity is quite similar and the endogenous modifications of HMb therefore have little effect on the reactivity of the protein intermediates.

NGB (human neuroglobin), a recently discovered haem protein of the globin family containing a six-co-ordinated haem, is expressed in nervous tissue, but the physiological function of NGB is currently unknown. As well as playing a role in neuronal O2 homoeostasis, NGB is thought to act as a scavenger of reactive species. In the present study, we report on the reactivity of metNGB (ferric-NGB), which accumulates in vivo as a result of the reaction of oxyNGB (oxygenated NGB) with NO, towards NO2- and H2O2. NO2- co-ordination of the haem group accounts for the activity of metNGB in the nitration of phenolic substrates. The two different metNGB forms, with and without the internal disulfide bond between Cys46 (seventh residue on the inter-helix region between helices C and D) and Cys55 (fifth residue on helix D), exhibit different reactivity, the former being more efficient in activating NO2-. The kinetics of the reactions, the NO2--binding studies and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate an active species with the chemical properties of peroxynitrite, at pathophysiological concentrations of NO2- and H2O2. Without external substrates, the targets of the reactive species generated by the metNGB/NO2-/H2O2 system are endogenous tyrosine (resulting in the production of 3-nitrotyrosine) and cysteine (oxidized to sulfinic acid and sulfonic acid) residues. These endogenous modifications were characterized by HPLC-MS/MS (tandem MS) analysis of metNGB after reaction with NO2- and H2O2 under various conditions. The internal S-S bond affects the functional properties of the protein. Therefore metNGB acts not only as scavenger of toxic species, but also as a target of the self-generated reactive species. Self-modification of the protein may be related to or inhibit its postulated neuroprotective activity.

The chemical conjugation of bisphosphonates (BPs), specifically alendronate, to hydroxyapatite could be an effective means to impart to it fine-tuned bioactivity. Horse heart myoglobin (Mb), a well-characterized protein, has been adsorbed onto biomimetic hydroxyapatite nanocrystals (nHA) and onto the nHA/alendronate conjugate powdered samples. The obtained materials have potential use in bone implantation and as prospective drug-delivery devices. The kinetic absorption of Mb onto nHA is dramatically affected by its functionalization with alendronate. The covering of the nHA surface by alendronate inhibits the adsorption of myoglobin. The adsorption mechanisms of the protein were studied by spectroscopic techniques (UV-vis and surface-enhanced Raman spectroscopy). The results indicate that the protein changes conformation upon adsorption on the inorganic substrate. In particular, the interaction with nHA alters the coordination state of the iron in the heme through the formation of a hexacoordinated low-spin Mb heme, possibly involving the distal histidine. Instead, the covering of the nHA surface by alendronate does not adsorb the protein but preserves the coordination state of the heme moiety. This study could be of significance either in the field of biomaterials science, in particular, to fine tune a bone-specific drug delivery device and to test nHA as a new support for heterogeneous catalysis, improving the understating of enzyme immobilization.