HEME AXIAL LIGATION BY THE HIGHLY CONSERVED HIS RESIDUES IN HELIX-II OF CYTOCHROME-B (NARI) OF ESCHERICHIA-COLI NITRATE REDUCTASE-A (NARGHI)

Optical spectroscopy and EPR studies confirm the existence of two b-ty pe hemes in the NarI subunit (cytochrome b(nr)) of the membrane-bound nitrate reductase (NarGHI) of Escherichia coli. Replacement of His-56 by Arg and His-66 by Tyr results in the loss of the high-potential hem e and of the low-potential heme, respectively. These data support the assignment of the axial ligands to the low-potential heme (His-66 and His-187) and to the high-potential heme (His-56 and His-205). This pai ring is consistent with the model proposed for NarI of the nitrate red uctase of Thiosphaera pantotropha (Berks, B. C., Page, M. D., Richards on, D. J., Reilly, A., Cavill, A., Outen, F., and Ferguson, S. J. (199 5) Mol. Microbiol. 15, 319-331) in which the two bis-histidine ligated hemes are coordinated by conserved His residues of helix II and V. EP R and optical studies suggest that the low-potential heme (E-m,E-7 = 17 mV) and the high-potential heme (E-m,E-7 = +122 mV) are located nea r the periplasmic side and the cytoplasmic side of the membrane, respe ctively. Moreover, correct insertion of both hemes into NarI requires anchoring to NarGH.