H-1-NMR OF HIGH-POTENTIAL IRON-SULFUR PROTEIN FROM THE PURPLE NONSULFUR BACTERIUM RHODOFERAX FERMENTANS

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPI P) from the purple non-sulfur photosynthetic bacterium Rhodoferax ferm entans have been characterized using H-1-NMR spectroscopy. Pairwise an d sequence-specific assignments of hyperfine-shifted H-1-NMR signals t o protons of cysteine residues bound to the [4Fe-4S](3+/2+) cluster ha ve been performed using one-dimensional NOE and exchange spectroscopy experiments. H-1-NMR hyperfine shifts and relaxation rates of cluster- bound Cys beta-CH2 protons indicate that in the [4Fe-4S](3+) cluster o ne iron ion can be formally described as Fe(III), while electron densi ty corresponding to one electron is unevenly delocalized onto the rema ining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C a lpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligeable contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting struc tural information from relaxation time measurements is proposed.