The reactivity of thiol compounds with different redox states of leghaemoglobin: evidence for competing reduction and addition pathways

Abstract

Reaction of the ferric form of leghaemoglobin with hydrogen peroxide has been previously shown to give rise to an iron(IV)-oxo (ferryl) species, and a protein radical. Inclusion of a variety of thiol compounds in this system is shown to lead to rapid loss of the iron(IV)-oxo species and the regeneration of the ferric form and/or the formation of novel sulf species formed by nucleophilic attack of the thiol group on the tetrapyrrole ring. The reduction process also results in the generation of thiyl radicals which have been detected by EPR spin trapping. The relative yields of the products produced by these two competing pathways is shown to be highly dependent on the steric and electronic characteristics of the thiol compound. Evidence has also been obtained, in the absence of hydrogen peroxide, for both the reduction of the ferric form of the protein to the oxy-ferrous form, via a process believed to involve the deoxy-ferrous species, and the formation of sulf-leghaemoglobin species. Both of these pathways are again highly dependent on the structure of the thiol, and the former also results in the generation of thiyl radicals. Inclusion of the sulfide anion in place of the organic thiols results in somewhat different behaviour, in that this species appears to both reduce the iron centre and form a complex with the iron atom. This ligation process is reversible, and the sulfide complex is shown to react readily with both strong oxidizing and reducing agents. The behaviour of this protein, which is structurally related to myoglobin, is dramatically different to that demonstrated by myoglobin; this is rationalized in terms of the much more open heme site of leghaemoglobins, and the presence of an electronic gate which hinders access by negatively charged molecules. The contribution of these processes to the maintenance of the leghaemoglobin proteins in the oxy-ferrous form in vivo and the binding of oxygen is discussed.