Epitopes of Monoclonal Antibodies Which Inhibit Ubiquinol Oxidase Activity of Escherichia coli Cytochrome d Complex Localize Functional

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases: the cytochrome d com- plex and the cytochrome o complex. Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molec- ular oxygen to water. Both oxidases are coupling sites in the respiratory chain; electron transfer from ubiquinol to oxygen results in the generation of a proton electrochemical potential difference across the mem- brane. The cytochrome d complex is a heterodimer (subunits I and II) that has three heme prosthetic groups. ubiquinol.

Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water. Both oxidases are coupling sites in the respiratory chain; electron transfer from ubiquinol to oxygen results in the generation of a proton electrochemical potential difference across the membrane.
The cytochrome d complex is a heterodimer (subunits I and II) that has three heme prosthetic groups. Previous studies characterized two monoclonal antibodies that bind to subunit I and specifically block the ability of the enzyme to oxidize ubiquinol. In this paper, the epitopes of both of these monoclonal antibodies have been mapped to within a single 11-amino acid stretch of subunit I. The epitope is located in a large hydrophilic loop between the fifth and sixth putative membrane-spanning segments. Binding experiments with these monoclonal antibodies show this polypeptide loop to be periplasmic. Such localization suggests that the loop may be close to His"', which has been identified as one of the axial ligands of cytochrome &s.
Together, these data begin to define a functional domain in which ubiquinol is oxidized near the periplasmic surface of the membrane.
The cytochrome d complex is one of the terminal oxidases in the aerobic respiratory chain of Escherichiu coli (see Ref. 1). Mutants of strains in which the cytochrome d complex is the only respiratory oxidase present in the membrane grow normally on nonfermentable substrates such as DL-lactate or succinate (2). The cytochrome d complex has been purified (3,4) and demonstrated to catalyze the two-electron oxidation of ubiquinol-8 within the bilayer (5) and the four-electron reduction of molecular oxygen to water (6). Reconstitution studies have shown that electron flow through this enzyme from ubiquinol to oxygen is electrogenic and generates a proton motive force (5, 7). Hence, the cytochrome d complex is a coupling site in the aerobic respiratory chain. The cytochrome d complex is a heterodimer, with one copy each of subunit I (58,000 Da) and subunit II (43,000 Da) (8). The enzyme contains three heme prosthetic groups: two protoheme IX and one heme d moieties (9-11). The two protoheme IX groups form two distinct cytochrome components: cytochrome bbb8 and cytochrome b5g5. Cytochrome bb58 appears to be a six-coordinate cytochrome that is known to be located entirely within subunit I (12, 13). Cytochrome b5g5, previously called cytochrome al, appears to be a five-coordinate, high spin heme, and its function is not known (9)(10)(11)(14)(15)(16). It may play a direct role in the reduction of oxygen to water. Cytochrome d is definitely involved in oxygen binding (10,(17)(18)(19)(20), and the heme d prosthetic group appears to be uniquely found in this enzyme (21).
Previous studies have strongly suggested that the catalytic active site at which ubiquinol oxidation occurs is spatially separate from the site at which oxygen is reduced (22). For example, trypsin proteolysis of the purified enzyme results in loss of ubiquinol oxidase activity but has no influence on the ability of the complex to oxidize the artificial electron donor N,N,N',N'-tetramethylphenylenediamine (22). Also, two monoclonal antibodies raised previously bind to the cytochrome d complex and inhibit ubiquinol oxidase activity without affecting the N,N,N',N'-tetramethylphenylenediamine oxidase activity of the enzyme (23). In each case, the target site was shown to be within subunit I.
The cyd operon, encoding both subunits of the enzyme, has been cloned (21) and sequenced (24). The deduced amino acid sequences suggest that both subunits I and II span the membrane, with seven and eight putative transmembrane segments, respectively (24). Some aspects of the topology of the subunits with respect to the bilayer have been elucidated using gene fusion techniques* (25). The monoclonal antibodies that are the subject of this study identify a hydrophilic loop involved in quinol oxidation and localize it on the periplasmic side of the bilayer.
The topological evidence plus the measured stoichiometry of proton translocation catalyzed in the enzyme (H+/e-P 1) (7) support a postulated model. It was proposed (22) that the enzyme functions by oxidizing ubiquinol at a site near the periplasmic surface, releasing two protons. The electrons are directed to a second site, located near the cytoplasmic surface, at which oxygen is reduced to water via a peroxy intermediate. The electron flow from the ubiquinol to the oxygen site generates a voltage across the membrane. The utilization of protons to form water on the cell interior and the release of protons as a result of ubiquinol oxidation near the periplasm are a scalar mechanism resulting in net proton flux.
The location of cytochrome bss, (E, s +180 mV) (9) on subunit I (12, 13) implicates this heme group in the oxidation of ubiquinol. This is consistent with the results of site-directed mutagenesis experiments in which Hisls6 within subunit, I was altered to a leucine (26). The mutant complex specifically lacks cytochrome bsss and is devoid of either ubiquinol or N, N,N',N'-tetramethylphenylenediamine oxidase activities.
His'% is probably located near the periplasmic surface of the membrane.
In the work described in this paper, the epitopes of the two inhibitory monoclonal antibodies have been mapped to the same 11-amino acid stretch within subunit I. These residues are located within a hydrophilic loop that is shown to be periplasmic and, therefore, very likely to be near to His"'. These data suggest the outlines of a domain within subunit I for the oxidation of ubiquinol.

MATERIALS AND METHODS
Strains-The tetracycline-sensitive E. coli strain GR84N. which lacks both subunits of the cytochrome d complex, was described previously (21). The E. coli strain Y1090 is a suppressor for the X mutation S7, and its use as a host for Xgtll has been described (29). The plasmid pNG2 contains the cyd gene and is used to overexpress the cytochrome d complex (21). The plasmid pFHlO1 also contains the cyd gene, but the vector allows for easier manipulation (26).

RESULTS
The sequences within the cyd gene which encode the epitopes for the monoclonal antibodies A14-5 and A16-1 were mapped by constructing and screening a Xgtll library containing small random fragments of the operon cyd. DNA fragments with random blunt end points were produced by digestion of pFHlO1 (26)  The epitope for the two inhibitory monoclonal antibodies is boxed on the large hydrophilic loop oriented toward the periplasmic side of the membrane.
Also indicated is the location of HisIS, which is an axial ligand of cytochrome bssa.  phage was produced. The cyd sublibrary was screened with each of the two monoclonal antibodies. Each revealed about 40 positive clones from approximately 5000 recombinant plaques screened. Of these, 20 plaques positive for each antibody were purified to homogeneity.
The DNA sequences that encode each epitope were determined using the dideoxy chain-termination method (Fig. 1). Single-stranded DNA primers complementary to Xgtll DNA sequences on either side of the EcoRI site were used to sequence past the junction into the insert DNA. Sequences of DNA insert end points were determined for 14 of the Xgtll subclones (Fig. 1). It became apparent that the epitopes for both monoclonal antibodies were very close, so phage positive for one antibody were tested for the ability to express both epitopes. All 14 phage with sequenced inserts were able to express the epitopes for both Al4-5 and A16-1. DNA sequence common to them all was deduced and shown to be 33 base pairs long. The amino acid sequence shown in Fig. 1 was expressed in frame in each case.
The location of the 11-amino acid stretch containing both epitopes is shown in the context of a tentative topological model of subunit I in Fig. 2.
Since the two monoclonal antibodies bind within a single short amino acid stretch, competition for binding would be expected. Fig. 3 shows the inhibition of '251-labeled A14-5 binding to cytochrome d complex by A16-1. Empirical constraints prevented determination of the A16-1 ratio at which maximal inhibition occurs. The appearance of inhibition after a lo-fold excess of A16-1 over A14-5 corresponds with the binding assay (Fig. 4), which shows A14-5 to have approximately lo-fold higher binding affinity.
The binding assay provides a good assessment of the binding affinity of A14-5 because radiolabeling allowed for direct quantitation of the ratio of antibody and ligand. Unfortunately, the amount of lz51-labeled Al6-1 was inadequate to achieve concentrations high enough to determine the full extent of its binding curve. Nevertheless, the onset of A16-1 binding shows it to be of substantially lower affinity than A14-5. '251-Labeled A14-5 was used to prove that the hydrophilic loop containing the epitope was directed toward the periplasm. Fig. 5 shows that A14-5 binds significantly more to spheroplasts of a strain expressing the cytochrome d complex than to inside-out vesicles of the same strain containing the same amount of cytochrome d. Spheroplasts were used instead of right-side-out vesicles because doubts exist regarding the stability and extent of their right-side-out orientation (32). The possibility of observing more counts with the spheroplasts due to higher nonspecific binding was controlled by the experiment quantifying binding to an equal amount of spheroplasts made from a strain lacking the cytochrome d complex.

DISCUSSION
The epitopes of the two inhibitory monoclonal antibodies have been clearly shown by this work to be within an llamino acid segment in subunit I. Some overlap of their individual epitopes is probable since the ability of the monoclonal antibody A14-5 to bind to the cytochrome d complex is inhibited by the presence of A16-1. Although gross steric hindrance may be responsible for this observation, the short length of the ll-amino acid stretch sufficient to bind either monoclonal antibody suggests otherwise. The mapping of the epitope within the primary sequence of subunit I has allowed determination of the position of an important hydrophilic loop with respect to the membrane bilayer. Through binding of the monoclonal antibodies to the cytochrome d complex in spheroplasts and vesicles, the large hydrophilic loop between the fifth and sixth putative membrane-spanning segments has been localized to the periplasmic side of the bilayer. Because the monoclonal antibodies that bind to this loop specifically inhibit ubiquinol oxidase activity, this loop will be referred to as the "Q-loop." Trypsin and chymotrypsin cleavage sites, which also cause specific inhibition of ubiquinol oxidase activity, have been mapped to the Q-loop within 30 amino acids of the monoclonal antibody epitope.3 The two-dimensional model of subunit I shown in Fig. 2 illustrates the location of the epitope in relation to the bilayer. It should be noted that the membrane-spanning regions shown are predicted from hydropathy profiles and therefore are not absolutely certain. However, it is evident that if the assignment of transmembrane segments is correct, the antibody-binding site lies on the same side of the membrane as His'*'. The strong possibility of close proximity between the epitope and His'% suggests that these residues are at or near the site at which ubiquinol is oxidized. Together, their location begins to define a functional domain for ubiquinol oxidation in the cytochrome d complex. At the very least, their location supports the postulate that ubiquinol is oxidized on the periplasmic side of the membrane and releases protons into the periplasm. 25.