Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands.

The heme protein subunit of sulfite reductase (SiR-HP; M(r) 64,000) from Escherichia coli as isolated contains the isobacteriochlorin siroheme exchange-coupled to a [4Fe-4S] cluster in the 2+ oxidation state. SiR-HP in the presence of a suitable electron donor can catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia. Paramagnetic 1H NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates sulfite and nitrite. As a model, the cyanide complex of purified siroheme was also prepared. The NMR spectrum of isolated ferric low-spin siroheme-CN is consistent with spin density being transferred into the a2u molecular orbital, an interaction which is symmetry-forbidden in porphyrins. The pattern of proton NMR shifts observed for isolated ferric low-spin siroheme-CN is very similar to those obtained for the protein-cyanide complex. NMR spectra of the cyanide complex of SiR-HP were obtained in all three accessible redox states. The pattern of hyperfine shifts observed for the one-electron and two-electron reduced cyanide complexes is typical of those seen for [4Fe-4S] clusters in the 2+ and 1+ oxidation states, respectively. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned for all complexes studied utilizing deuterium substitution. The cyanide-, sulfite-, and nitrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coupling exists between siroheme and cluster in solution. Data are also presented for the existence of a secondary anion binding site, the occupancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than that of ligand binding to heme.