Assignment of the Histidine Axial Ligands to the Cytochrome b_H and Cytochrome b_L 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, His198, 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 His198 by Asn or Tyr resulted in loss of both cytochrome components. However, His111 Asn, His111Asp, and His212Asp 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 Hisl98). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane α-helices.