Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria

Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) is composed of hemoprotein and flavoprotein subunits. The heme prosthetic group, responsible for interactions of the enzyme with sulfite, is a novel octacarboxylate iron-tetrahydroporphyrin of the isobacteriochlorin type, which has been given the name siroheme. Rate constants for the formation (kon) of E. coli sulfite reductase-CO complex and E. coli sulfite reductase hemoprotein-CO complex have been measured. The kon for the native reduced enzyme and its hemoprotein subunit had values of 6.9 m-1 s-1 and 7.7 m-1 s-1, respectively. Such kon values are extremely slow when compared to those of other hemoproteins such as hemoglobin and cytochrome oxidase. However, the isolated siroheme prosthetic group, in the absence of protein, exhibited a kon for CO complex formation of 1 x 107 m-1 s-1, a value comparable to those found for other free hemes; the siroheme-CO complex was photodissociable. No dissociation of the holoenzyme or hemoprotein-CO complexes could be detected under reducing conditions. A very slow dissociation (koff = 6.2 x 10-5 s-1 for holoenzyme-CO and 9.2 x 10-5 s-1 for hemoprotein-CO) of CO from its complexes with enzyme could be detected aerobically, with formation of oxidized heme-enzyme. In contrast to siroheme-CO, no photodissociation of holoenzyme-CO complex was detected. It is concluded that the rates of association and dissociation of carbon monoxide with sulfite reductase reflect profound hindering effects exerted on the heme by its immediate environment in the hemoprotein subunit, and are not due to constraints imposed by either the flavoprotein portion of the holoenzyme or by the unusual structure of the iron-tetrahydroporphyrin itself.