ASSIGNMENT OF THE HISTIDINE AXIAL LIGANDS TO THE CYTOCHROME-BH AND CYTOCHROME-BL COMPONENTS OF THE BC1 COMPLEX FROM RHODOBACTER-SPHAEROIDES BY SITE-DIRECTED MUTAGENESIS

The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome b(H) and cytochrome b(L) Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, Hisl98, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of Hisl98 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His2l2Asp all resulted in the selective loss of cytochrome b(H) and the retention of cytochrome b(L). Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Q(z)) site associated with cytochrome b(L) is still functional. These data support the assignment of the axial ligands to cytochrome b(H) (His111 and His212) and cytochrome b(L) (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane a-helices.