Exploring Subunit-Subunit Interactions in the Escherichia coli bo-type Ubiquinol Oxidase by Extragenic Suppressor Mutation Analysis*

(Received for publication, March 4, 1997, and in revised form, March 27, 1997)

Keitarou Saiki , Tatsushi Mogi , Motonari Tsubaki Dagger , Hiroshi Hori § and Yasuhiro Anraku

From the Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, the Dagger  Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-12, and the § Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cytochrome bo-type ubiquinol oxidase is a four-subunit heme-copper terminal oxidase and functions as a redox-coupled proton pump in the aerobic respiratory chain of Escherichia coli. On the basis of deletion and chemical cross-linking analyses on subunit IV, we proposed that subunit IV is essential for CuB binding to subunit I and that it is present in a cleft between subunits I and III (Saiki, K., Nakamura, H., Mogi, T., and Anraku, Y. (1996) J. Biol. Chem. 271, 15336-15340).

To extend previous studies, we carried out alanine-scanning mutagenesis for selected 16-amino acid residues in subunit IV to explore subunit-subunit interactions in bo-type ubiquinol oxidase. We found that only the replacement of Phe83 in helix III resulted in the reduction of the catalytic activity but that this did not significantly affect the UV-visible spectroscopic properties and the copper content. This suggests that individual amino acid substitutions, including the six invariant residues, are not enough to alter such properties of the metal centers. Extragenic suppressor mutations were isolated for the Phe83 right-arrow Ala mutation of subunit IV and identified as missense mutations in helices VII and VIII in subunit I. These observations provide further support for specific interactions of subunit IV with helix VII and/or VIII, the CuB binding domain, of subunit I and suggest that subunit IV functions as a domain-specific molecular chaperon in the oxidase complex.


INTRODUCTION

In the aerobic respiratory chain of Escherichia coli, cytochrome bo-type ubiquinol oxidase (UQO)1 is predominantly expressed under highly aerobic growth conditions (1) and functions as a redox-coupled proton pump (2). UQO consists of four different subunits (3, 4) that are encoded by the cyoABCDE operon (5). Subunits I, II, and III (CyoB, CyoA, and CyoC, respectively) of UQO are homologous to the counterparts of mitochondrial and bacterial cytochrome c oxidases, and thus UQO belongs to the heme-copper terminal oxidase superfamily (5, 6). Subunit I binds all three redox metal centers, low spin heme b (cytochrome b563.5), high spin heme o (cytochrome o), and a copper ion (CuB) (7-11). The latter two metal centers form the heme-copper binuclear center that serves as the catalytic center for dioxygen reduction and proton pumping. Site-directed mutagenesis studies demonstrated that six invariant histidines in subunit I serve as the axial ligand of the metal centers and that a bundle of helices VI-VIII and X form a binding pocket for the binuclear center. Subunit II does not have any metal center but seems to provide the ubiquinol oxidation site (12). Subunits III and IV can be removed from the UQO complex without loss of the catalytic activity (13); however, they are essential for the functional expression of the UQO complex (14).2 Subunit IV (CyoD), whose homologues can be found only in the bacterial terminal oxidases (5, 14), is proposed to be located in a cleft between subunits I and III and to assist the CuB binding to subunit I (14). Deletion analysis of CyoD revealed that the functional domain is located in the C-terminal two-thirds (Val45-His109) containing helices II and III (14) (see Fig. 1). The cyoE gene, which presents at the 3'-end of the cyo operon, encodes heme O synthase, which supplies heme O specifically to the binuclear center of UQO (16).


Fig. 1. Topological model of subunit IV (CyoD) of bo-type ubiquinol oxidase from E. coli (modified from Chepuri and Gennis (15)). Amino acid residues conserved in six out of eight subunit IV sequences of the bacterial heme-copper terminal oxidases (5, 14, 21) are shown in the circles. Phe83, whose substitution by analine reduced the catalytic activity, is indicated by the diamond.
[View Larger Version of this Image (28K GIF file)]

To extend previous studies on subunit IV (14), we carried out alanine-scanning mutagenesis to identify the functional amino acid residues that are involved in interactions between subunits I and IV. Subsequently, we isolated and characterized extragenic suppressor mutations for the defective subunit IV mutation, namely D-F83A.3 We found that extragenic mutations are located in transmembrane helices VII and VIII of subunit I. Thus, we conclude that subunit IV is in close vicinity to the CuB binding site of subunit I and functions as a domain-specific molecular chaperon in the UQO complex.


MATERIALS AND METHODS

Bacterial Strains, Plasmids, Growth Media, and DNA Manipulations

E. coli strains, plasmids, and growth media used in this study and DNA manipulations were as described previously (8, 14, 17). Ampicillin was supplemented to growth media at a final concentration of 15 µg/ml for mini-F plasmids or 50 µg/ml for multicopy plasmids.

Construction of the CyoD Mutants

Alanine-scanning mutagenesis of CyoD was carried out using phagemid pCYOF6 and mutagenic primers in the range of 21-30 nucleotides (14). The 0.4-kb EcoRI-EagI or the 0.4-kb EagI-EcoRV fragment containing the cyoD mutations was isolated from phagemid DNAs and replaced with the counterpart in the wild-type multicopy plasmid pCYO6 (14). The nucleotide sequence of the corresponding region in the recombinant plasmid was confirmed by direct plasmid sequencing (8). For expression of the mutant enzymes, the EcoRI-SphI fragment of the mutant pCYO6 was introduced into the corresponding sites of mini-F plasmid, pMFO21 (14). To facilitate the subcloning of the entire cyo coding region, a single copy expression vector pHNFO11P, which carried the gene-engineered unique ClaI and BamHI sites before and after the cyo promoter, respectively, was constructed from pMFO1 (8). Similarly, a multicopy expression vector pBR4 was constructed from pHN3795-1 (18). pHNFO11P-O9B and pBR4-O9B are the derivatives of pHNFO11P and pBR4, respectively, and contain the whole cyo operon.

Isolation of Extragenic Suppressor Mutants for the D-F83A Mutation

ST2592 (Delta cyo Delta cyd)/pBR4-O9B-DF83A, which had been anaerobically grown overnight in 2 × YT medium supplemented with 0.5% NaNO3, was inoculated into 5 ml of minimal medium A (19) containing 0.5% glycerol, 0.5% glycerol plus 0.05% glucose, or 0.5% glucose and then cultured aerobically at 37 °C for 3 days. Cultures with significant turbidity were streaked on minimal/0.5% glycerol plates, and revertants were isolated after incubation at 37 °C for 2 days. Assignment of the DNA segments carrying the suppressor mutations was carried out by subcloning of the restriction fragments of plasmid DNA isolated from revertant strains into pBR4-O9B-DF83A. Recombinant plasmids were then introduced into ST2592 to confirm the ability of transformants to grow aerobically on minimal/glycerol plates. The identified DNA fragments were subcloned into pUC119 for DNA sequencing analysis with a model 373A DNA sequencer (Applied Biosystems Inc.).

Construction of His-tagged CyoD

pCYO64-DHisCt was constructed by cloning of a synthetic His5 tag linker at the 3'-end of the cyoD gene using the genetically engineered unique SplI site in pCYO64 (17). Then, the 1.7-kb EcoRI-SphI fragment of pCYO64-DHisCt was subcloned into pHNF11OP-O9B to give pHNFO11P-O9B-DHisCt.

Isolation of Cytoplasmic Membrane Vesicles

Cytoplasmic membranes were isolated from ST4676 (Delta cyo cyd+) harboring the pMFO11P-O9B or pBR4-O9B derivatives (14, 17). A large scale isolation of the membranes was performed as described previously (20).

Purification of UQO

The membranes were diluted to a protein concentration of 1.5 mg/ml with 50 mM Tris-HCl (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 0.5 M NaCl, and 1.5% sucrose monolaurate SM-1200 (SM) (Mitsubishi-kasei Food Co., Tokyo) and solubilized with stirring at 4 °C for 1 h. The mixture was centrifuged at 180,000 × g for 1 h, and all (mutants) or half (wild type) of the supernatant was passed twice through 12 ml of Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen Inc.) at a flow rate of ~6 ml/min. After washing Ni-NTA-agarose resins with 150 ml of wash buffer (50 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride, 0.5 M NaCl, and 0.1% SM), UQO was eluted by 30 ml of wash buffer containing 0.1 M imidazole. Peak fractions were desalted and concentrated by ultrafiltration using a Macrosep 100 (Filtron Technology). Contaminated phospholipids were removed by anion-exchange chromatography (20) after a 20-fold dilution with 50 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride and 1% SM.

Miscellaneous

Other analytical procedures were as described previously (8, 13, 17). Restriction endonucleases and other enzymes for DNA manipulations were purchased from Takara Shuzo Co. (Kyoto, Japan) or New England BioLabs, Inc. Other chemicals were commercial products of analytical grade.


RESULTS

Alanine-scanning Mutagenesis of CyoD

Subunit IV (CyoD) of the UQO complex is composed of 109 amino acid residues and has three transmembrane helices (I-III) with the N terminus in the cytoplasm (Refs. 5 and 15; Fig. 1). Homologues of CyoD are found only in bacterial quinol and cytochrome c oxidases (5, 14, 21). Among eight known subunit IV sequences, 17-51% of the amino acid residues are conserved and Phe22, Leu24, Leu28, and Thr29 in helix I, Ala54 and Gln57 in helix II, and Phe65, His67, and Met68 in the cytoplasmic loop II-III are found to be invariant and appear to be essential for the UQO function. However, our recent deletion analysis demonstrated that the N-terminal cytoplasmic domain, helix I, and the loop I-II are dispensable in CyoD (14). Subunit IV seems to be present in a cleft between subunits I and III of the UQO complex (14), as found in a crystal structure for bacterial aa3-type cytochrome c oxidase (22). To explore subunit-subunit interactions in the heme-copper terminal oxidases, we extended the previous studies on subunit IV and carried out alanine-scanning mutagenesis on 12 conserved amino acid residues and 4 aromatic or charged amino acid residues within or at the boundary of transmembrane helices of subunit IV (Fig. 1, Table I).

Table I. Characterizations of the CyoD alanine mutants in cytoplasmic membranes

The in vivo catalytic activity of the mutant oxidases was evaluated by the genetic complementation test using ST2592 harboring pMFO1 derivatives. The amounts of cytochromes (Cyt) o and b563.5 were determined by CO binding difference spectra and 77 K redox difference spectra, respectively. Contents of copper ions were estimated by atomic absorption spectroscopy. Expression level of subunit I was examined by Western blotting using anti-subunit I antiserum followed by densitometric analysis (8). Strains ST4676 harboring pMFO1 (cyo+) and pHNF2 were used as the wild-type and the negative controls, respectively.

Mutant Catalytic activity Cyt o Cu Cyt b563.5 Subunit I

nmol/mg protein nmol/mg protein
Wild type + 0.39 0.33 +++ +++
Control  - 0.05 0.01  -  -
 Delta CyoD (Delta D5)a  - 0.26 0.01 ++ +++
D-Y18A + 0.32 0.30 +++ +++
D-F22A + 0.32 0.31 +++ ++
D-T29A + 0.30 0.38 +++ +++
D-F33A + 0.41 0.44 +++ +++
D-W34A + 0.30 0.30 +++ ++
D-Q57A + 0.32 0.29 +++ +++
D-H61A + 0.27 0.30 +++ +++
D-F65A + 0.35 0.38 +++ +++
D-H67A + 0.29 0.31 +++ +++
D-M68A + 0.30 0.35 +++ +++
D-K71A + 0.37 0.48 +++ +++
D-E74A + 0.33 0.33 +++ +++
D-F81A + 0.31 0.30 +++ +++
D-F83A  - 0.29 0.28 ++ +++
D-W97A + 0.35 0.22 +++ +++
D-W100A + 0.29 0.41 +++ +++

a Data taken from Ref. 14.

Catalytic activities of the mutated oxidases were examined by genetic complementation test (Table I). Using a single copy vector pMFO21, the cyoD mutant operons were expressed in a terminal oxidase-deficient strain ST2592 that cannot grow aerobically on nonfermentable carbon sources via oxidative phosphorylation. The wild-type and all the CyoD alanine mutants except D-F83A grew aerobically on minimal/0.5% glycerol plates, whereas the D-F83A mutant and Delta D5 mutant, which carries the complete cyoD deletion (14), as well as the vector control strain ST2592/pHNF2 (8) failed to grow aerobically (Table I). This result indicates that alanine can be substituted for the 15 amino acid residues examined in the present study without a significant loss of the UQO functions. To test the gene dosage effect, we overexpressed the D-F83A mutant UQO in ST2592/pHN3795-1-DF83A and found that it can grow slowly on minimal/glycerol plates (Table II), indicating that the D-F83A mutant oxidase is partially functional.

Table II. Characterizations of the extragenic suppressor mutants for the D-F83A mutation

The in vivo catalytic activity of the mutant oxidases was evaluated by the genetic complementation test using ST2592 harboring a multicopy vector (pBR4-O9B or pHN3795-1 derivatives). The cytoplasmic membranes were prepared from ST4676 (Delta cyo cyd+) harboring pHNFO11P-O9B derivatives and used for UV-visible spectroscopic and Western blotting analyses. Other conditions are described in the legend to Table I.

Mutant Catalytic activity
Cyta o Cyt b563.5 Subunit I
pBR4-09B pHN3795-1

nmol/mg
Wild type + + 0.39 +++ +++
 Delta cyoD (Delta D5)  -  - 0.29 ++ +++
D-F83A  - ± 0.29 ++ +++
D-F83A/B-L326P + + 0.29 ++ +++
D-F83A/B-I357T + + 0.27 ++ +++
D-F83A/B-V361M + + 0.34 +++ +++
B-L326P + + 0.25 + +++
B-I357T + + 0.25 + +++
B-V361M + + 0.32 ++ +++

a Cyt, cytochrome.

Characterizations of the CyoD Alanine Mutant UQOs

Effects of the alanine substitutions in CyoD on the redox metal centers were examined using cytoplasmic membrane vesicles isolated from strain ST4676 (Delta cyo cyd+) harboring the pMFO1 derivatives. Low spin heme b was quantitated as cytochrome b563.5 (8, 20) by an amplitude of the 563.5-nm peak in the second order finite difference spectra of redox difference spectra at 77 K. The content of high spin heme o was estimated as cytochrome o (20) from CO binding difference spectra at room temperature. The CuB content was estimated as the amount of bound copper ions in the membranes (8, 20). As shown in Table I, all of the mutations in CyoD did not largely alter the amounts of high spin and low spin heme signals and CuB. The amounts of the three metal centers in the D-F83A mutant were reduced to 80% that of the wild-type levels, whereas the defective CyoD deletions completely lost the CuB center (14).

Isolation of Extragenic Suppressor Mutations for the D-F83A Mutation

To identify a defect(s) caused by the D-F83A mutation, we isolated extragenic suppressor mutants based on their ability to grow aerobically on minimal/glycerol medium. To facilitate genetic manipulations of the large cyo operon (i.e. 5 kb), we constructed a multicopy expression vector pBR4-O9B where the 2.2-kb 5'- and the 2.5-kb 3'-noncoding regions of the cyo operon in pHN3795-1 (18) have been deleted, but it contained five genetically engineered restriction sites (NheI, ApaI, XhoI, MluI, and Bsu36I)4 in the structure genes and ClaI and BamHI sites around the cyo promoter (Fig. 2). Spectroscopic analysis of the cytoplasmic membranes showed that the expression level of UQO in strain ST4676 by multicopy vector pBR4-O9B was slightly less than those by single copy vectors pMFO1 and pHNFO11P-O9B (~85%; Tables I and II) although the generation time of ST2592/pBR4-O9B in minimal/glycerol medium was nearly identical to that of ST2592/pHNFO11P-O9B (data not shown). Accordingly, the ST2592/pBR4-O9B-DF83A mutant was also unable to grow aerobically in minimal/glycerol medium (Table II).


Fig. 2. Locations of extragenic suppressor mutations for the D-F83A mutation. a, the physical map of the cyoABCDE operon. The minimal DNA regions carrying four extragenic suppressor mutations are indicated by horizontal bars. The genetically engineered restriction sites present in mini-F plasmid pHNF11P-O9B are marked by asterisks. b, the secondary structure model of subunit I. Putative transmembrane helices (5, 15) are indicated by boxes.
[View Larger Version of this Image (24K GIF file)]

We isolated four spontaneous revertants independently from strain ST2592/pBR4-O9B-DF83A that had been grown in minimal medium containing 0.5% glycerol plus 0.05% glucose (R1, R3, and R4) or minimal/0.5% glycerol medium (R2) (Table II). Plasmid DNAs isolated from the revertants were digested with convenient restriction enzymes as indicated in Fig. 2a, and the resultant fragments were subcloned into the corresponding sites of pBR4-O9B-DF83A to test the ability of recombinant plasmids to support the aerobic growth of ST2592 on minimal/glycerol plates. After several rounds of screening, the suppressor mutation of the R1 revertant was mapped in the 0.14-kb XhoI-MluI fragment and those of the R2, R3, and R4 revertants were mapped in the 0.29-kb MluI-Bsu36I fragment (Fig. 2a). Sequencing analysis showed that all the suppressor mutations were extragenic missense mutations in the subunit I (cyoB) gene as follows. A codon for Leu326 (CTG) was changed to Pro (CCG) in R1 (B-L326P), that for Ile357 (ATC) to Thr (ACC) in R2 (B-I357T), and that for Val361 (GTG) to Met (ATG) in R3 and R4 (B-V361M) (Fig. 2b). Leu326 is located in transmembrane helix VII where His333 and His334 ligate CuB at the periplasmic end (9-12). Ile357 and Val361 are present in helix VIII, which also consists of a binding pocket for the heme-copper binuclear center (8-11). These results indicate the presence of specific interactions of subunit IV with transmembrane helices VII and/or VIII of subunit I in the UQO complex.

Effects of Suppressor Mutations on the Metal Centers

The suppressor mutations were subcloned into pHNFO11P-O9B, and their effects on the high spin and low spin hemes were examined in the presence and the absence of the original D-F83A mutation. Western blotting analysis of the cytoplasmic membranes indicated that all of the mutations did not affect the stability of the mutant UQOs (Table II). UV-visible spectroscopic analysis of the membranes showed that reductions in the amounts of cytochromes o and b563.5 by the D-F83A mutation were partially suppressed by the B-V361M whereas all three individual suppressor mutations significantly reduced heme signals although they did not affect the aerobic growth of the mutant strains (Table II).

Characterizations of the Purified Mutant UQOs

The mutant UQOs were purified by Ni-NTA-agarose chromatography using the His6 tag including His109 at the C terminus of CyoD (D-HisCt). The purity of the isolated D-HisCt UQO from the small scale culture was estimated to be ~90%. Unexpectedly, we found that the wild-type and mutant UQOs without the His tag can be purified similarly to the His-tagged UQO (Fig. 3). Upon the addition of the His tag, the apparent molecular mass of subunit IV increased from 13.5 to 14.5 kDa in 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (lanes 1 and 2). Alternatively, the complete CyoD deletion eliminated a 13.5-kDa polypeptide (lane 4), providing further support for identification of subunit IV as CyoD. It should be noted that a 7-kDa polypeptide seems to be associated with the UQO complex even after DEAE-5PW HPLC (lanes 5-9) and size exclusion HPLC (data not shown). It can be seen in the UQO preparation obtained by anion-exchange HPLC (20) when the gel was carefully fixed (data not shown). In contrast, subunit V (20) is almost absent in the UQO preparation with the small scale protocol (lanes 1-4) as compared with the large scale protocol (lanes 5, 7, and 8). These results indicate that subunit IV is not required for the assembly of the UQO complex and that the mutations in subunits I (B-L326P, B-I357T, and B-L361M) and IV (D-F83A) do not disrupt the quaternary structure of the UQO complex.


Fig. 3. SDS-polyacrylamide gel electrophoresis analysis of the purified mutant UQOs. UQOs were purified by Ni-NTA affinity chromatography. Small scale purification (lanes 1-4) was performed by incubating 1 mg of solubilized membrane proteins with 50 µl of Ni-NTA-agarose, and large scale purification (lanes 5-9) was done at the 1.8-g scale followed by anion-exchange HPLC. The D-HisCt UQO was purified from the membranes of ST2592/pHN3795-1-DHisCt (lane 1). The others were purified from the membranes of ST4676 harboring the pHNFO11P-O9B derivatives: lanes 2 and 5, wild type; lanes 3 and 6, D-F83A; lane 4, Delta CyoD (Delta D5); lane 7, D-F83A/B-L326P; lane 8, D-F83A/B-I357T; and lane 9; D-F83A/B-V361M mutants. Aliquots (0.25 µg) of the purified enzymes were subjected to 14% SDS-polyacrylamide gel electrophoresis. Proteins were visualized by silver staining and calibrated with Rainbow Protein Molecular Weight Markers (Amersham Life Science, Inc.). Subunits of the UQO complex are indicated by roman numerals.
[View Larger Version of this Image (73K GIF file)]

The effects of the D-F83A and suppressor mutations on the metal centers and the catalytic activity were further studied using the purified enzyme obtained with the large scale protocol. The isolated UQOs were estimated to be ~70% pure based on their heme contents (Fig. 4, Table III). As expected from the complementation test (Table II), the D-F83A mutation reduced ubiquinol-1 oxidase activity to 55% that of the wild-type control (Table III). The B-I357T and B-V361M subunit I mutations completely suppressed the defects caused by the D-F83A mutation in subunit IV, whereas the B-L326P mutation reduced CO binding activity of high spin heme o and quinol oxidase activity to 79 and 41%, respectively, that of the wild-type levels (Fig. 4, Table III). The alpha  peak of pyridine ferrohemochrome from the mutant UQOs was found at 554.5 nm, the same as the wild-type enzyme that binds 1 mol each of hemes B and O (20), suggesting that these mutations did not alter the specificity of the heme binding sites. In addition, the amount of cytochrome b563.5 remained unaltered (Fig. 4, Table III), indicating that a defect is distal to the low spin heme binding site. These results suggest that the defect of the D-F83A mutation cannot be attributed to a loss of any metal centers.


Fig. 4. Second order finite difference spectra of dithionite-reduced minus air-oxidized difference spectra (a) and CO-reduced minus reduced difference spectra (b) of the purified mutant UQOs. a, spectra were recorded with a Shimadzu UV-3000 double wavelength spectrophotometer at 77 K with a spectral bandwidth of 1 nm and a light path of 2 mm. Scan rate was 50 nm/min, and protein concentrations were 1.0 mg of protein/ml of 120 mM Tris-HCl (pH 7.4) containing 0.1% SM. b, conditions used were the same as those described for a except that the measurements were carried out at room temperature at a protein concentration of 0.2 mg of protein/ml with a light path of 10 mm.
[View Larger Version of this Image (26K GIF file)]

Table III. Characterization of the purified D-F83A extragenic suppressor mutant enzymes

The mutant UQOs were purified by large scale protocol. Heme contents are expressed as the sum of protoheme IX and heme O (20). Ubiquinol oxidase activity was measured as described (14) except that a molar extinction coefficient for ubiquinone-1 of 12,300 was used. The parentheses indicate a percentage for the wild-type value referred to as 100%. Other details are described in the legends to Fig. 3 and Table I.

Mutant Cyta o Cu Heme Ubiquinol oxidase

nmol/mg protein e-/sec
Wild type 3.8  (100)b 4.5  (100)b 8.2 505  (100)b
D-F83A 4.0  (105) 4.6  (102) 8.8 280  (55)
D-F83A/B-L326P 3.0  (79) 6.8  (151) 8.2 206  (41)
D-F83A/B-I357T 3.8  (100) 5.6  (124) 8.2 533  (105)
D-F83A/B-V361M 4.0  (105) 4.7  (104) 8.2 480  (95)
D-HisCt 4.1  (108) NDc 8.2 419  (83)

a Cyt, cytochrome.
b % wild-type value.
c Not determined.


DISCUSSION

Recently, the crystal structures of aa3-type cytochrome c oxidases have been solved at atomic resolution for the 4-subunit enzyme from Paracoccus denitrificans (22) and for the 13-subunit enzyme from bovine heart (23). Coordination chemistry of the redox metal centers revealed by molecular biological studies on bacterial enzymes (8-11) was confirmed by crystallographic efforts (22, 23). This structural information provides us with new insights into the molecular mechanism of redox-coupled proton pumping. Subunits I and II are known to serve as the redox reaction centers in the oxidase complex, whereas the functional role of other small subunits remains obscured. The nuclear encoded "supernumeral" subunits of eukaryotic oxidases are unlikely to be involved in electron transfer or proton pumping (23) but may serve as regulators of the cytochrome c oxidase activity through ATP binding and/or the switching of tissue-specific isoforms (24). Subunit III of bacterial cytochrome c oxidase has been suggested to be required for assembly of subunits I and II into an active complex (25).

Comparison of the bacterial subunit IV sequences indicates that helix I to the loop II/III is more conserved than the other portions; however, our deletion analysis of CyoD showed that the N-terminal one-third (i.e. the N terminus to the loop I/II) of subunit IV is dispensable in the catalytic function of UQO (14). CyoD was suggested to assist the binding of copper ions to the CuB site during the folding process of subunit I or assembly of the UQO complex (14). Alanine-scanning mutagenesis demonstrated that only a substitution of Phe83 in helix III reduced the catalytic activity without significant effects on the redox metal centers and the assembly of the UQO complex. Since Phe or Tyr is conserved at position 83, aromatic side chains at the cytoplasmic end of helix III may be crucial for folding of subunit IV or interaction with subunit I. Aromatic side chains on intrinsic membrane proteins are frequently found within membrane boundaries (26) and may be stabilized by the choline head group of phospholipids through cation-pi interactions (27). Individual replacements of other conserved amino acid residues in subunit IV were not enough to alter subunit-subunit interactions; therefore, the functional role of subunit IV seems to be structural rather than enzymatic in contrast to CyoE (16, 17).

Recently, Ni-NTA affinity chromatography was shown to be efficient for small scale purification to near homogeneity of the His-tagged UQO and cytochrome c oxidase from Rhodobacter sphaeroides (28)5 and may be suitable for isolation of unstable mutant enzymes. UQO with the His-tagged CyoD was purified to ~90 and 70% using small scale and large scale protocols, respectively (Fig. 3, Table III). However, it should be noted that an endogenous cation binding site in the C-terminal periplasmic domain of subunit II (29) seems as efficient as the His tag introduced at the C terminus of CyoD (Fig. 3). Purification of the CyoD-deficient mutant oxidase by this method demonstrated that subunit IV is not required for the assembly of subunits I-III (Delta CyoD (Delta D5) in Fig. 3, lane 4). Based on the deletion and chemical cross-linking analyses on CyoD, we proposed previously that subunit IV is located in a cleft between subunits I and III (14), similar to the four-subunit cytochrome c oxidase of P. denitrificans (22). Specific interactions of helices II and III of subunit IV with subunit I has been suggested from a complete loss of the CuB center in the CyoD deletion mutants (14). In the present study, we identified the extragenic suppressor mutations for the D-F83A mutation in helices VII and VIII of subunit I (B-L326P, B-I357T, and B-V361M). It has been shown previously that helices VI, VII, and VIII of subunit I form the CuB binding pocket and contain the axial ligands for CuB (His284, His333, and His334) (8-11). These results suggest the presence of direct interactions of subunit IV with subunit I.

In conclusion, subunit IV is dispensable for enzyme activity (13) but is required for assembly of the oxidase complex into a catalytically active form through interactions with the CuB binding domain of subunit I.


FOOTNOTES

*   This work was supported in part by Grants-in-aid for Scientific Research on Priority Areas 08249106 and 08268216 to (T. M.), for Scientific Research 07558221, 07309006, and 08458202 to (T. M.), and for Exploratory Research 08878097 to (T. M.) from the Ministry of Education, Science, Sports, and Culture, Japan. This is paper XXIII in the series "Structure-Function Studies on the E. coli Cytochrome bo Complex."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Fax: 81-3-3812-4929.
1   The abbreviations used are: UQO, cytochrome bo-type ubiquinol oxidase from E. coli; kb, kilobase pair(s); SM, sucrose monolaurate SM-1200; Ni-NTA, Ni-nitrilotriacetic acid; HPLC, high performance liquid chromatography.
2   H. Nakamura, K. Saiki, T. Mogi, and Y. Anraku, unpublished data.
3   The designations for mutations make use of the standard one-letter abbreviations for amino acids. Thus, "D-F83A" signifies the mutant in which the phenylalanine at position 83 in CyoD (subunit IV) has been replaced by alanine.
4   J. Minagawa, T. Mogi, and Y. Anraku, unpublished data.
5   Recently, Gennis and co-workers demonstrated that Ni-NTA affinity chromatography was suitable for rapid purification of UQO when a His6 tag was placed at the C terminus of subunits I, II, and III (J. N. Rumbley, E. F. Nickels, and R. B. Gennis, unpublished data).

ACKNOWLEDGEMENTS

We thank H. Nakamura of Riken for valuable comments and R. B. Gennis of the University of Illinois for unpublished observations.


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