Jeffrey P. Osborne

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I

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.

DETERGENT-SOLUBILIZED ESCHERICHIA COLI CYTOCHROME BO3 UBIQUINOL OXIDASE : A MONOMERIC, NOT A DIMERIC COMPLEX

The protein molecular weight, M r, and hydrodynamic radius, R s, of Triton X‐100‐solubilized Escherichia coli cytochrome bo 3 were evaluated by computer fitting of sedimentation velocity data with finite element solutions to the Lamm equation. Detergent‐solubilized cytochrome bo 3 sediments as a homogeneous species with an s 20,w of 6.75 s and a D 20,w of 3.71×10−7 cm2/s, corresponding to a R s of 5.8 nm and a M r of 144 000±3500. The protein molecular weight agrees very well with the value of 143 929 calculated from the four known subunit sequences and the value of 143 025 measured by MALDI mass spectrometry for the histidine‐tagged enzyme. We conclude that detergent‐solubilized E. coli ubiquinol oxidase is a monomeric complex of the four known subunits.

Rapid Formation of a Semiquinone Species on Oxidation of Quinol by the Cytochrome bo3 Oxidase from Escherichia coli

Many bacterial oxidases utilize dihydroquinols, such as ubiquinol or menaquinol, rather than cytochrome c as a substrate. The best-characterized ubiquinol oxidase is cytochrome bo 3 from Escherichia coli. In this work, the initial oxidation of ubiquinol by this ubiquinol oxidase is examined. Stopped-flow UV-visible spectroscopy and rapid freeze-quench electron paramagnetic resonance (EPR) spectroscopies were used to examine the oxidation of ubiquinol-2 (UQ2H2) by cytochrome bo 3 under multiple turnover conditions. The results show the rapid appearance of the semiquinone radical, coincident with the reduction of the low-spin heme b component of the enzyme. The rate of formation of the semiquinone radical is consistent with the proposition that this is a kinetically relevant intermediate in the reaction sequence. As UQ2H2 is depleted, the radical decays and the enzyme forms a “peroxy,” or P, complex with dioxygen. No detectable protein radical is associated with the P complex.