Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria: II. IDENTIFICATION OF A NEW CLASS OF HEME PROSTHETIC GROUP: AN IRON-TETRAHYDROPORPHYRIN (ISOBACTERIOCHLORIN TYPE) WITH EIGHT CARBOXYLIC ACID GROUPS

Abstract

The novel heme prosthetic group of Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) has been identified as an iron tetrahydroporphyrin of the isobacteriochlorin type (adjacent pyrrole rings reduced) with eight carboxylic acid side chains. The number of carboxyl groups was established by the following experiments. Esterification with 14CH3OH resulted in incorporation of 7.4 moles of 14C per mole of heme. The fragmentation pattern of the demetallized heme (porphin) methyl ester and a comparison of the heaviest mass peaks of the porphin ethyl and methyl esters was consistent with the presence of eight carboxylate ester side chains. The porphin methyl ester migrated similarly to uroporphyrin I octamethyl ester in two thin layer chromatographic systems. The reduction state of the porphin nucleus was determined as follows. The infrared spectrum of the porphin ethyl ester showed a band at 1603 cm-1, characteristic of reduced porphins. The absorption spectra of the porphin ester (λmax (ratios) = 378 (3.3), 510 (0.37), 545 (0.67), 588 (1.00), 638 (0.15) nm) in neutral or basic media and its tin, copper, and zinc derivatives were similar to those of synthetic isobacteriochlorins and their corresponding metal derivatives. The porphin in neutral solvents exhibited an intense orange fluorescence (λmax = 597 and 640 nm for emission) indicating the presence of a cyclically conjugated porphin macrocycle. The tin porphin ester was not photoreduced under conditions which led to conversion of tin uroporphyrin octamethyl ester to the tetrahydro derivative. Precise molecular masses of several derivatives of the sulfite reductase porphin yielded a unique empirical formula for each derivative; these formulas, when restricted to a porphin with eight carboxylate side chains, require the porphin nucleus to be at the tetrahydro or more reduced level. No formulas compatible with octacarboxylate porphyrins or chlorins could be obtained. The empirical formula for the sulfite reductase heme is FeC42H44N4O16 (z ligands not included). It is suggested that the compound is an iron-containing methylated urotetrahydroporphyrin.