Optical spectroscopy and EPR studies confirm the existence of two b-type hemes in the NarI subunit (cytochrome bnr) 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 heme 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 pairing is consistent with the model proposed for NarI of the nitrate reductase of Thiosphaera pantotropha (Berks, B. C., Page, M. D., Richardson, D. J. , Reilly, A., Cavill, A., Outen, F., and Ferguson, S. J. (1995) Mol. Microbiol. 15, 319-331) in which the two bis-histidine ligated hemes are coordinated by conserved His residues of helix II and V. EPR and optical studies suggest that the low-potential heme (Em,7 = +17 mV) and the high-potential heme (Em,7 = +122 mV) are located near the periplasmic side and the cytoplasmic side of the membrane, respectively. Moreover, correct insertion of both hemes into NarI requires anchoring to NarGH.