Mechanism of a novel synthesis of haemin c from protohaemin and L-cysteine. A Markownikoff-type radical addition reaction

Abstract

As a simulation of in vivo sulphide bond formation of c-type cytochromes, haemin c(4) was synthesized by the reaction of iron protoporphyrin IX [protohaemin (3)] with sodium borohydride in the presence of L-cysteine, oxygen, and cetyltrimethylammonium bromide (CTAB). When L-cysteine was omitted from the reaction mixture, mesohaemin (9) and haematohaemin (5) were obtained. The inhibitory effect of cyanide anion or carbon monoxide, as well as the inability of protoporphyrin (8) to serve in place of (3) in both reactions, indicated that the iron of (3) and oxygen were crucial in a primary process to give a common intermediate (11) for (4), (5), and (9). Trapping of (11) with oxygen indicated that it is the α-carbon radical of the 2- or 4-ethyl group derived from (3). The addition of deuterium (from sodium borodeuteride) to the β-carbon of the vinyl group of (3) and the resulting formation of (11) strongly suggested the intermediacy of a free hydrogen atom, which was generated in the reduction of (3) with sodium borohydride. The generation of a free hydrogen atom was also supported by a transfer experiment.