The Main Role of Inner Histidines in the Molecular Mechanism of Myoglobin Oxidation Catalyzed by Copper Compounds

Abstract

In the presence of Cu(2+) and Cu(Gly)(2), the oxidation of two native MbO(2)'s (Mb = myoglobin), from the sperm whale and horse, and also two chemically modified sperm whale MbO(2)'s alkylated at solvent-accessible histidines by sodium bromoacetate (CM-MbO(2)) and by iodoacetamide (CA-MbO(2)) have been studied at different pH's, ionic strengths, and concentrations of the copper reagent. The influence of competitive redox-inactive zinc ions on the reaction rate is investigated as well. Localization of Cu(Gly)(2) in sperm whale met-Mb and CM-met-Mb has been examined using the high-resolution NMR method. The obtained data suggest that binding of copper compounds to the surface histidines (all of them are 1.8-2.7 nm apart from the heme) has only a minor, no more than 35%, contribution to the overall reaction rate, in particular under a large excess of the reagent (more than 8-10-fold). The noticeable contribution of His113(116), His48, and His81, which have the greatest affinity to copper according to NMR data, is revealed only at small concentrations of copper, less than a 5-fold excess relative to the protein. The main contribution to the reaction rate must be from the binding of copper to the inner histidines, His97 (0.62 nm from the heme), and possibly to the distal His64. Both are inaccessible to the modification by alkylating reagents and have much lower affinity to copper than all surface histidines, because they are hydrogen-bonded, the former with the carboxyl group of the heme propionate and the second with the liganded O(2).

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

“…Early His modifications were achieved by using bromoacetic acid and bromoacetate. ,, Two nitrogen atoms on the imidazole are potential nucleophilic sites for modification. As halogen atoms are good leaving groups, many α-halo carboxylic acids and amides were developed and utilized in His labeling, including but not limited to bromoacetate, ,− chloroacetate, ,, iodoacetate, ,, bromoacetamide, and IAM . Although effective, these reagents soon fell into disuse, primarily due to their lack in specificity.…”

“…IAM today is one of the most widely used Cys alkylating reagent as mentioned in section . His labeling by bromoacetate, a reagent that can be used for Cys-free proteins, however, requires days to complete, greatly limiting broad application , and causing concerns about structural change during the footprinting and misleading overlabeling. A similar situation applies to methyl- p -nitrobenzenesulfonate, which can label His residues but now principally serves as a alkylation reagent for Cys. , …”