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) fr om Escherichia coli as isolated contains the isobacteriochlorin sirohe me exchange-coupled to a [4Fe-4S] cluster in the 2+ oxidation state. S iR-HP in the presence of a suitable electron donor can catalyze the si x-electron reductions of sulfite to sulfide and nitrite to ammonia. Pa ramagnetic H-1 NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates su lfite and nitrite. As a model, the cyanide complex of purified sirohem e was also prepared. The NMR spectrum of isolated ferric low-spin siro heme-CN is consistent with spin density being transferred into the a2u molecular orbital, an interaction which is symmetry-forbidden in porp hyrins. 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 ob tained 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-C H2 protons of cluster cysteines have been assigned for all complexes s tudied utilizing deuterium substitution. The cyanide-, sulfite-, and n itrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coup ling exists between siroheme and cluster in solution. Data are also pr esented for the existence of a secondary anion binding site, the occup ancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than that of ligand binding to heme.