The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b558. At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231-239] according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595.

Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.

Electron nuclear double resonance (ENDOR) was performed on the protein-bound, stabilized, high-affinity ubisemiquinone radical, QH*-, of bo3 quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings. Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool. In contrast, QH*- serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal bo3 oxidase. As documented by large hyperfine couplings (>10 MHz) to nonexchangeable protons on the QH*- ubisemiquinone ring, we provide evidence for an electronic distribution on QH*- that is different from that of the semiquinones of reaction centers. Since the ubisemiquinone itself is physically nearly identical in both QH*- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment. Interaction of QH*- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix.