One-electron reduction reactions with enzymes in solution. A pulse radiolysis study

Abstract

At pH 8 and above, hydrated electrons react with ribonuclease, lysozyme and α-chymotrypsin to form transient products whose spectra resemble, but are not identical to, those for the RSSR^– radical anion already known for simple disulphides. Assuming a value for the extinction coefficient similar to that for RSSR^– in simple disulphides, only a fraction of the hydrated electrons are shown to react with the disulphide bridges: the remainder react at other sites in the protein molecule, such as histidine, tyrosine and, in lysozyme, tryptophan residues, giving rise to comparatively weak optical absorptions between 300 and 400 nm. This has been substantiated by studying the reaction of e^–_aq with subtilisin Novo(an enzyme which does not contain disulphide bridges), with enzymes in which the sulphur bridges have been oxidised and with some amino acid derivatives.

On lowering the pH of the solution the intensity of the RSSR^– absorption diminishes as the protonated histidine residues become the favoured reaction sites. In acid solutions (pH 2–3) the transient optical absorptions observed are due to reactions of hydrogen atoms with the aromatic amino acids tyrosine, tryptophan and phenylalanine.

The CO^–₂ radical anion is only observed to transfer an electron to disulphide groups in ribonuclease, although the effect of repeated pulsing shows that some reaction must occur elsewhere in the protein molecule. In acid solutions, protonation of the electron adduct appears to produce the RSSRH˙ radical, whose spectrum has a maximum at 340 nm.