Pseudomonas pseudoalcaligenes KF707 grown with biphenyl expresses a cytochrome caa₃ oxidase that uses cytochrome c₄ as electron donor

Abstract

Combining peroxidase activity based haem-staining (TMBZ/SDS-PAGE) with mass spectrometry analyses (Nano LC-MS/MS) of protein extracts from wild type and appropriate mutants, we provide evidence that the polychlorinated biphenyl (PCB) degrader Pseudomonas pseudoalcaligenes KF707 primarily expresses a caa₃-type cytochrome c oxidase (caa₃-Cox) using cytochrome (cyt) c₄ as an electron donor in cells grown with biphenyl versus glucose as the sole carbon source. Homology modeling of KF707 caa₃-Cox using the three-dimensional structure of that from Thermus thermophiles highlights multiple similarities and differences between the proton channels in subunit I of the aa₃- and caa₃-Cox of Paracoccus and Thermus spp., respectively. To our knowledge, this is the first report demonstrating the presence of a caa₃-Cox using cyt c₄ as an electron donor in a Pseudomonas species.

1. Introduction

Cytochrome (cyt) c oxidases (or cyt c: O₂ reductases) belong to the haem copper oxidase (HCO) superfamily [1]. HCO members are classified into three main families, type A, B, and C, based on the sequence signatures and conservation of their proton transport pathways (named D and K). Among the type A HCO, the mitochondrial cyt c oxidases (Cox) and their closely related counterparts from bacteria such as Paracoccus denitrificans and Rhodobacter sphaeroides, are referred to as aa₃-Cox [2]. These enzymes contain a low-spin a-type haem and a haem a₃-Cu_B binuclear center at their catalytic subunit (SU) I, as well as a haem binuclear Cu_A center at their SU II. Electrons are received initially from cyt c by the Cu_A center, and conveyed via haem a to the haem a₃-Cu_B center where O₂ is converted to H₂O [1]. According to the amino acid motifs found at the hydrophobic end of the D-proton pathway, the type A HCOs can be further divided into two subclasses: type A1 contains a highly conserved glutamate residue in the sequence motif – XGHPEV – whereas in type A2 the gating glutamate is replaced by tyrosine - serine in the motif – YSHPXV [3]. The type B HCOs are found in bacteria and archaea, and contain a low-spin b-type haem and a high-spin haem a (ba₃-Cox). The type C HCOs are quite different from both type A and B Cox with respect to their amino acid and subunit compositions, and contain three c-type haems and two b-type haems (cbb₃-Cox) [4].

The first crystal structure of a type A2 HCO was described from the thermophilic Gram-negative bacterium Thermus thermophilus [5]. This HCO consists of two SUs, referred to as SU I/III and SU IIc that contain all of the metal centers characteristics of aa₃-Cox [6]. In this enzyme, the SU I/III is a fusion of the classical SU I and SU III whereas SU IIc is a fusion between a canonical SU II and a cyt c domain. Due to this feature, the HCO of T. thermophilus is also called caa₃-Cox. This type of HCO is present in the thermophilic Gram negative Rhodothermus marinus and the Gram-positive Bacillus subtilis species [3,7]. The occurrence of a cyt c domain fused with a canonical SU II seems to reflect the need for a better control of the respiratory electron transfer under harsh environmental conditions (e.g., high temperatures), or in the absence of a periplasmic space in Gram positive species [1,8]. Similar to aa₃-Cox, the proton pumping stoichiometry of caa₃-Cox (0.8–1 H⁺/e⁻) is higher than ba₃- (0.5–0.75 H⁺/e⁻) and cbb₃- (0.2–0.5 H⁺/e⁻) Cox, also present in thermophilic and mesophilic bacteria [3,9,10,44].

Recently, genome analyses of the polychlorinated biphenyl (PCBs) degrader Pseudomonas pseudoalcaligenes KF707 (hereafter called KF707) suggested the presence of a gene cluster (BAU71738-71737-71740) encoding an aa₃-Cox composed of three subunits (SU I, SU IIc and SU III) [11]. The predicted SU IIc (42 kDa) of KF707 was annotated as a membrane-anchored cupredoxin domain containing one electron-accepting homo-binuclear copper center Cu_A, with a carboxyl terminal fusion to a cyt c domain [5,11]. The occurrence of a c-type haem in the KF707 aa₃-Cox was also suggested by sequence alignments which showed, at the C-terminal region, the typical amino acid residues (CXXCH) that coordinate the c-type haem in B. subtilis and T. thermophilus caa₃-Cox [5,8] (Figure 1). These haem binding residues are not found in the aa₃-Cox from R. sphaeroides and P. denitrificans. Notably, the SU IIc amino acid sequence of KF707 shows 81% of identity with CoxB (SU II) of the opportunistic pathogen Pseudomonas aeruginosa PAO1 [11,12]. In this latter species, the presence of an orthodox aa₃-Cox was documented by Arai and co-workers [13,14,15], although this group referred recently to the product of P. aeruginosa PAO1 cox genes [16] as caa₃-Cox despite any supporting data.

Figure 1.

A. Organization of the Caa₃-Cox gene cluster in KF707: dark gray, functional genes (coxIIc-I-III); light gray, genes for Caa₃-Cox biogenesis and white, genes probably not directly related to Caa₃-Cox. B. Alignment analysis for subunit II of the caa₃-type Cox from KF707 (Pp - BAU71737), T. thermophilus (Tt – AFH37994), P. aeruginosa (Pa – ONM68414) and B. subtilis (Bs – OBA08775), and aa₃-type Cox from R. sphaeroides (Rs – ASN35548). The first alignment focuses on the cupredoxin domain and the copper center Cu_A (N-terminal portion), and the second one shows the cytochrome c domain (C-terminal portion), absent in the aa₃-type Cox from R. sphaeroides. The dark boxes are the amino acids that are directly involved in the Cu_A and haem c binding, asterisks and dots represent the fully and partially conserved amino acid residues, respectively. Analyses were performed using the ClustalW software [20].

Our studies aimed at understanding the metabolic abilities of KF707 in using biphenyl as a sole carbon source [17,18], indicated that membranes from KF707 cells grown with different carbon sources (e.g., glucose or biphenyl) contained several membrane-bound c-type haem proteins. In the present study, using tetramethylbenzidine (TMBZ) staining of SDS-PAGE and tandem mass spectrometry (MS) analyses, we defined the identities of these hemoproteins, and demonstrated that the protein band of 37 kDa corresponded to the c-type cyt domain containing SU IIc of KF707 caa₃-Cox. As expected, this 37 kDa band was absent in a KF707 mutant lacking cox1-2 gene clusters (KFΔcox1-2, Caa₃- and Ccaa₃-minus). We also showed that this SU IIc is highly expressed in membranes from cells grown in the presence of biphenyl, while it is poorly expressed in those grown in the presence of glucose. This finding is consistent with our recent study related to the promoter activity of the cox1 (caa₃-Cox) gene cluster [11]. To further support our findings, a homology based structural model of KF707 caa₃-Cox was built using the X-ray structure of that from T. thermophilus (PDB code 2YEV) [5]. Finally, the TMBZ/SDS-PAGE, mass spectrometry and ascorbate/TMPD oxidase activity measurements using membranes from a KF707 triple mutant (KFΔc₄/cco1-2), which lacks the di-haem cyt c₄ (BAU71765) and the cbb₃-Cox-1 and -Cox-2, grown in biphenyl showed that cyt c₄ is the electron donor to caa₃-Cox. To the best of our knowledge, this work represents the first report that unequivocally demonstrates the presence in a Pseudomonas species of a caa₃-Cox that uses cyt c₄ as an electron donor.

2. Material and Methods

2.1. Bacterial strains and construction of terminal oxidase deleted mutants

The bacterial strains used are listed in Table 1, and Pseudomonas pseudoalcaligenes KF707 was used as a wild type strain (W.T.). Nucleotide and amino acid sequences were from the complete genome sequence of KF707 available in GenBank (accession number AP014862). Sequence similarity searches were performed using the BLAST software (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) [19] and the conserved domain database (http://www.ncbi.nlm.nih.gov/structure/cdd/), and multiple sequence alignments were performed with ClustalW software [20]. Single and multiple Cox-deletion mutants of KF707, Gene SOEing PCR [21], pG19II conjugative plasmid [22] and double crossover recombination were used as described previously [11]. Deletions of the cyt c₄ (BAU71765) or cyt c₅ (BAU71764) genes were constructed using appropriate primers listed in Table S3.

Table 1.

List of the Pseudomonas pseudoalcaligenes KF707 strains used in this study.

Bacterial strains	Relevant Genotype
Wild type (W.T.)	Ampr
KFΔcco1-2	Cbb31 and Cbb32 minus - Deletion of ccoN1O1Q1P1 and ccoN2O2Q2P2, Ampr
KFΔcox1-2	Caa3 and Ccaa3 minus - Deletion of coxI-IIc-III and coxMNOP, Ampr
KFΔcox1-2/cco1-2	Caa3, Ccaa3, Cbb31 and Cbb32 minus - Deletion of coxI-IIc-III, coxMNOP, coN1O1Q1P1 and ccoN2O2Q2P2, Ampr
KFΔCIO	CIO minus - Deletion of cioABC, Ampr
KFΔcox1-2/CIO	Cbb31, Cbb32 and CIO minus - Deletion of coxI-IIc-III, coxMNOP and cioABC, Ampr
KFΔc4	cyt c4 minus – Deletion of c4 (BAU71765), Ampr
KFΔc5	cyt c4 minus – Deletion of c4 (BAU71764), Ampr
KFΔc4/c5	cyt c4 and cyt c5 minus – Deletion of c4 (BAU71764) and c5 (BAU71764), Ampr
KFΔc4/cco1-2	cyt c4, Cbb31 and Cbb32 minus – Deletion of c4 (BAU71765), coN1O1Q1P1 and ccoN2O2Q2P2, Ampr

2.2. Cell membrane fragments preparation, protein concentration and respiratory activities determinations

For preparation of cell membranes, KF707 W.T. and mutant strains were grown aerobically at 30°C and 130 rpm in 3 L Erlenmeyer flasks containing 1 L Mineral Salt Medium (MSM), pH 7.2 [18], supplemented with 6 mM glucose or biphenyl as the sole carbon source. Cells were harvested at stationary phase (OD_{600 nm} 0.7–0.9), after 16 or 30 hours growth for glucose or biphenyl, respectively. Membrane fragments were prepared as previously described [23]. Protein concentrations were determined using the bicinchoninic (BCA) acid assay according to the supplier’s recommendations (Sigma Inc.; procedure TPRO-562). Respiratory activities in membrane fragments from appropriate strains were determined as reported previously [11].

SDS-PAGE and haem staining

The c-type cyt detection was performed using membrane fragments from KF707 W.T. and mutant strains grown in MSM medium with glucose or biphenyl. 150–200 μg of protein samples were denatured for 5 minutes at 37°C and separated by 15% SDS-PAGE [24]. The c-type cyts were revealed via their intrinsic peroxidase activity by using 3,3′,5,5′-tetramethylbenzidine (TMBZ) and H₂O₂ [25]. ImageJ served to quantify the intensity of the bands.

2.3. NADI staining

Cox phenotypes of colonies from KF707 W.T. and deletion mutants were determined using NADI staining, which visualizes their Cox activities. This staining is based on the oxidation of α-naphthol to indophenol blue via the artificial electron donor N,N,dimethyl-p-phenylenediamine (DMPD), coupled to Cox activity. NADI⁻negative indicates the absence, and NADI-positive (formation of blue staining) the presence of Cox activity in colonies [11].

2.4. Nano LC-MS/MS analysis and database searches

SDS-PAGE bands were excised, and after reduction (dithiothreitol, Sigma) and alkylation (iodoacetamide, Bio-Rad), subjected to in-gel trypsin digestion (Promega, Sequencing Grade Modified Trypsin) overnight at 37°C. Peptides eluted from the gel samples were dried and desalted using ZipTips (Milipore U-C18 P10), lyophilized and stored at −80 °C. They were resuspended in 10 uL 5% acetonitrile/0.1% formic acid prior to mass spectrometry, using Q-Exactive Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to an Easy-nLC^TM 1000 nano liquid chromatography system (Thermo Fisher Scientific, San Jose, CA). Samples were loaded in buffer A (0.1% formic acid) onto a 20 cm long fused silica capillary column (75 μm ID), packed with reversed-phase Repro-Sil Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Germany). Peptides were eluted using a 45 min linear gradient from 4%–40% buffer B (100% acetonitrile, 0.1% formic acid), followed by a 7 min gradient from 40%–80% buffer B and 8 min wash with 80% B (constant flow rate 300 nL/min). The Q-Exactive was operated in data-dependent acquisition (DDA) mode with dynamic exclusion enabled (repeat count: 1, exclusion duration: 20 sec). Full MS survey scans (mass range 300–1600 m/z) at high resolution (70,000 at 200 m/z) were followed by MS/MS fragmentation of the top 15 most intense ions with higher energy collisional dissociation (HCD) at a normalized collision energy of 22 (resolution 17,500 at 200 m/z). Dual lock mass calibration was enabled with 371.101233 and 445.120024 m/z background ions. MS spectra were searched against the KF707 protein database (UniProtKB) using Proteome Discoverer 1.4 Software (Thermo) with Sequest-HT search engine. Search parameters were set to full trypsin digestion, with a maximum of 3 missed cleavages and 3 modifications per peptide. Oxidation of methionine (+16 Da) and carbamido-methylation of cysteine (+57 Da) were selected as dynamic modifications. Precursor and fragment ion tolerances were set to 10 ppm and 0.6 Da, respectively. False discovery rates by target-decoy search (FDR) were set to 0.01 (high confidence) with X_corr of >2 for m/z=2, >2.5 for m/z=3, and > 2.6 for m/z=4 values.

2.5. Membrane protein modeling

Template searches for each of the three subunits (SU I, IIc, and III) of KF707 caa₃-Cox (Pp-caa₃) were performed using the HHsearch method implemented in the HHpred server [26]. The server performs up to eight iterative PSI-BLAST [27] searches through filtered versions of the non-redundant (nr) database from NCBI. Using the final target alignment, a hidden Markov model (HMM) [28,29] profile is calculated. Homologous templates are identified by searching through a database containing HMMs for a representative subset of PDB sequences. HHsearch ranks the database matches based on the probability of the match to be homologous to the target sequence to distinguish homologous from non-homologous matches. Excluding PDB structures that were not solved by X-ray crystallography, HHpred identified the caa₃-Cox from T. thermophilus (Tt-caa₃). Tt-caa₃ is composed of two proteins homologous to Pp-caa₃ SU I/III and SU IIc sequences. SU I/III is a fusion of the canonical SU I to SU III, connected with a linker of ca. 70 residues. The pronounced sequence identity/similarity of Pp-caa₃ SU I, IIc and III with respect to the corresponding sequences of Tt-caa₃ (45/62%, 30/45% and 31/42%, respectively) allows the generation of a reliable structural model. The target and template sequences were realigned using the Promals3D server [30] (see Supplementary Information for the alignments). The sequences of aa₃-Cox subunit II and III from P. denitrificans (Pd-aa₃) were included to align two large insertions not present in the Tt-caa₃ sequences. The obtained alignment was then used to calculate 100 structures using the available crystal structures of Tt-caa₃ (2YEV) [5] and Pd-aa₃ (3HB3 [31] and 1QLE [32]) to model Pp-caa₃ subunit IIc and III, respectively, using the Modeller 9.18 software [33]. The two haems a, the haem c, the binuclear and mononuclear copper centers (Cu_A and Cu_B, respectively) and the Mg(II) ion, together with the water molecules in direct contact with the metal ions and the lipid molecules found in the Tt-caa₃ structure, were included in the modeling procedure. The best model was selected using the DOPE potential function built into Modeller [34]. A loop optimization routine was used to refine the regions that showed higher than average energy as calculated using the DOPE potential function. The stereo-chemical quality of the model structure was established using ProCheck [35] to confirm the reliability of the model structure. The obtained molecular model and its molecular surface were displayed using UCSF Chimera [36] and APBS [37] softwares. The position of the protein model in the membrane was predicted using the PPM web server [38], and the conservation of the surface residues was evaluated using the ConSurf 2016 server [39].

3. Results

3.1. Cyt c profile and Nano LC-MS/MS analysis of KF707 W.T. and appropriate mutants

Recently, we reported KF707 mutants carrying deletions of single or multiple terminal oxidases [11], which were crucial in understanding the unusual metabolic abilities of a polychlorinated biphenyls degrader such as KF707 [17]. Membranes from KF707 cells, grown with glucose or biphenyl as a carbon source, expressed several membrane-bound c-type cyts, corresponding to caa₃- and cbb₃-Cox. Genetic and functional analyses identified five terminal oxidases in KF707: two c(c)aa₃-Cox (Caa₃ and Ccaa₃), two cbb₃-Cox (Cbb₃1 and Cbb₃2) and one bd-type cyanide-insensitive quinol oxidase (CIO). While the expression of both cbb₃-Cox was prevalent in glucose grown cells, the promoter activity of the caa₃-Cox genes increased considerably when biphenyl was used as a single carbon source, whereas the promoter activity of cco2 gene corresponding to Cbb₃2 was repressed [11].

Presently, no biochemical or structural data are available to evidence the presence of any caa₃-Cox in the Pseudomonas species [1,40]. In order to fill this knowledge-gap, the cyt c contents of membranes isolated from KF707 W.T. and the Cox-deletion mutants were examined using TMBZ/SDS-PAGE [24,25]. Proteins were extracted from membrane fragments of cells harvested at their stationary phase of growth (OD_600nm 0.8–1.0) in MSM glucose or biphenyl (6 mM each). In membranes from glucose grown cells at least six TMBZ stained bands were found (Figure 2A, left), while those grown in biphenyl exhibited five such bands (Figure 2A, right). The lowest MW (~17 kDa) band, present in membranes of all strains grown on either carbon source (Figure 2A and B) was identified by MS analysis to be the cyt c₄ (see Section 3.3 and Table 2). Similarly, the two bands of 20–22 kDa were assigned to the CcoO1 and CcoO2 subunits of Cbb₃1 and Cbb₃2, respectively (Table S1), whereas in cells grown with biphenyl only the band related to CcoO1 was detected. In both glucose and biphenyl grown cells, a band attributable to cyt c₁ subunit (29 kDa) (Table S1) of the bc₁ complex of KF707 was also present [11]. Of the haem c protein bands with MW between 34–36 kDa, two in glucose (Figure 2A, left) and one in biphenyl (Figure 2A, right) were identified as the CcoP subunits of Cbb₃1 (CcoP1, 35.5 kDa) and Cbb₃2 (CcoP2, 34 kDa), respectively (Table S1). As expected, both bands were undetectable in membranes from the KFΔcco1-2 mutant, lacking the Cbb₃1 and Cbb₃2. Moreover, while the CcoP1 band was present in both glucose and biphenyl grown cells, the CcoP2 band was absent in biphenyl grown cells (Figure 2A, right). These findings were in line with previous data showing that the Cbb₃2 genes were not expressed in the stationary phase of growth when biphenyl was the sole carbon source [11].

Figure 2.

Profiles of the c-type cytochromes detected in membranes of KF707 W.T. and mutant strains grown with glucose or biphenyl as the carbon source until stationary phase (OD_{600 nm} 0.8–1.0). Membrane fragments were analyzed by SDS-PAGE and the c-type cytochromes were revealed via their peroxidase activities using TMBZ staining.

A – Membranes of cells grown in glucose (left) or biphenyl (right) are shown. Lane 1 and lane 7 on the left and the right gels contain the Precision Plus Protein Standards Ladder (BIO-RAD) molecular weight [MW] standards; the remaining lanes correspond to KF707 W.T., KFΔCIO, KFΔcox1-2, KFΔcco1-2, KFΔcox1-2/CIO and KFΔcox1-2/cco1-2, as indicated.

B – Bands of proteins containing cytochrome c domains with MW between 25 and 37 kDa, are shown in detail. KF707 W.T. cells grown with glucose was compared with other KF707 strains grown with biphenyl. Lane 1 contains Precision Plus Protein Standards Ladder (BIO-RAD) MW standards, lane 2 contains KF707 W.T. grown with glucose, Lane 3 to 8 correspond to KF707 W.T., KFΔCIO, KFΔcox1-2, KFΔcco1-2, KFΔcox1-2/CIO and KFΔcox1-2/cco1-2, respectively Mutant phenotypes are listed in Table 1. All experiments were repeated at least three times.

Table 2.

Nano LC-MS/MS analysis results of the 17 and the 37 kDa bands extracted from TMBZ gels of KF707 W.T. and mutant strains grown in MSM medium with glucose or biphenyl as the sole carbon source. The amino acids coverage, significant peptides and their modifications are listed.

17 kDa Glucose or Biphenyl	Accession	Description
	L8MT74	Cytochrome c4 OS=Pseudomonas pseudoalcaligenes KF707 = NBRC 110670 GN=ppKF707_770 PE=3 SV=1 - [L8Mt74_PSEPS] – NCBI Cyt c4 BAU71765 – Coverage 31,4 %
	Sequence	Modifications
	DIEAVSSYIQGLH
	AAVcGAcHGPDGNSAAPNFPK	C4-C7(Carbamidomethyl)
	TVLEmTGLLTNmSDQDmADLAAYFASQK	M5-M12-M17(Oxidation)
37 kDa Glucose	Accession	Description
	L8MR96	Cytochrome c oxidase subunit 2 OS=Pseudomonas pseudoalcaligenes KF707 = NBRC 110670 GN=ppKF707_5503 PE=3 SV=1 - [L8MR96_PSEPS] – NCBI CoxII BAU71737 – Coverage 32,4 %
	Sequence	Modifications
	APKDEHYLLEVDQPLVVPVGTK
	ELTDKEWTLDELVAR
	DEHYLLEVDQPLVVPVGTK
	DHGFmPIVVEAK	M5(Oxidation)
	YLGQDVEFFSNLATPSEQIHNK
	ADHLNIVFHGKPGTSmAAFGK	M16(Oxidation)
	DAIPGFVNESWTR
	NAWGNNTGDmVTPK	M10(Oxidation)
	Accession	Description
37 kDa Biphenyl
	L8MR96	Cytochrome c oxidase subunit 2 OS=Pseudomonas pseudoalcaligenes KF707 = NBRC 110670 GN=ppKF707_5503 PE=3 SV=1 - [L8MR96_PSEPS] – NCBI CoxII BAU71737 – Coverage 38,7 %
	Sequence	Modifications
	YLGQDVEFFSNLATPSEQIHNK
	ADHLNIVFHGKPGTSmAAFGK	M16(Oxidation)
	DEHYLLEVDQPLVVPVGTK
	ELTDKEWTLDELVAR
	ADHLNIVFHGKPGTSMAAFGK
	APKDEHYLLEVDQPLVVPVGTK
	KDAIPGFVNESWTR
	LKELTDKEWTLDELVAR
	GQcTELcGKDHGFmPIVVEAK	C3-C7(Carbamidomethyl); M14(Oxidation)
	DHGFMPIVVEAK
	DHGFmPIVVEAK	M5(Oxidation)
	NAWGNNTGDmVTPK	M10(Oxidation)
	EVLALKQAESQ
	EWTLDELVAR
	GQcTELcGK	C3-C7(Carbamidomethyl)

Notably, the ~ 37 kDa protein band seen in membranes from both glucose and biphenyl grown cells, was not present in mutants lacking the caa₃-Cox (KFΔcox1-2, KFΔcox1-2/CIO, KFΔcox1-2/cco1-2), and was roughly seven times more abundant in cells grown on biphenyl (Figure 2B) than those on glucose (Materials and Methods). Similarly, this 37 kDa was roughly eight time less abundant in glucose grown cells (Figure 2A and B) as compared with the 34 or the 35.5 kDa bands (CcoP2 or CcoP1 subunits), and about two-three times less than CcoP1 present in biphenyl grown cells (Figure 2A and B). These experiments were repeated four times, and the intensities of the bands evaluated by ImageJ (6.8 ± 0.7 biphenyl versus glucose).

The 37 kDa band was excised from gels run with glucose or biphenyl grown cells membranes, and subjected to MS analysis, which identified over one hundred co-migrating proteins. CoxII (Uniprot code L8MR96 – 42 kDa) was present among these proteins and contained the canonical haem binding CXXCH motif of a c-type cyt, as previously seen with the CoxB subunit of T. thermophilus Caa₃ [5]. In both cases, MS data identified several distinct peptides (8 for glucose and 15 for biphenyl grown cells) of the CoxII subunit, with an overall coverage of over 30% (Table 2, Figure 3A and B, Figure S1). These results strongly support the earlier conclusion made on the role of caa₃-Cox in biphenyl grown cells of KF707 based on the expression of its gene cluster [11].

Figure 3.

Examples of two of Nano LC-MS/MS fragmentation spectra obtained with the 37 kDa band from cells grown with glucose (A) or biphenyl (B). These bands were identified as the subunit SU IIc of KF707 caa₃-Cox.

3.2. Protein modeling of the caa₃-Cox

A model structure of KF707 caa₃-Cox from (Pp-caa₃), based on the X-ray structure of that from T. thermophilus (Tt-caa₃, 2YEV) was generated [5]. Pp-caa₃ consists of three subunits (I, IIc, and III) (Figure 4A). Its SU I is composed of 12 transmembrane α-helices connected to each other by unstructured linkers, and contains two a-type haem groups located in the hydrophobic core, and one mononuclear copper center (named Cu_B) (Figure 4A, B and C). SU IIc is a fusion protein between a canonical SU II and a cyt c domain, similar to that from T. thermophilus [41]. It is composed of two transmembrane α-helices in direct contact with SU I and a periplasmic domain consisting of a 7-strands antiparallel β-sheet fused to a typical cyt c protein. Hence, SU IIc contains a haem c group with the Fe-ion bound to its His282 and Met332, and a homobinuclear copper center (Cu_A) bound to its His181, Cys216, Glu218, Cys220, His224, and Met227 residues (Figure 4A and B). The SU III is composed of six transmembrane α-helices and is in direct contact with SU I.

Figure 4.

Ribbon representation of the caa₃-type Cox homology model of KF707; SU I, SU IIc and SU III are shown as single entities (A) or together (B) and with their cofactors (C). The subunits are colored light blue, yellow and light green in the in the proximity of the N-terminus, and dark blue, dark red and dark green at the C-terminus of SU I, IIc and III, respectively. In this KF707 caa₃-Cox model, Trp120 of Pp-caa₃ is highlighted since it substitutes for the Tt-caa₃ Phe126, which has similar electron transfer properties, while the copper binding Cys224 (Pp-caa₃ numbering) is not indicated as it is conserved in both sequences. The haems are shown as ball and stick with the iron and copper metal centers as orange and ochre spheres, respectively. The Mg(II) ion in SU I is shown as a green sphere. Residues involved in electron transport are reported as sticks, and colored accordingly to atom type.

The protein surface of Pp-caa₃ shows the typical charge distribution of a membrane protein (Figure S2), with a large hydrophobic region corresponding to the transmembrane portion of SU I, IIc and III, twined with electrostatically charged surfaces exposed to the aqueous environment of the periplasm and the cytoplasm. The protein surface of the membrane-extern portion of Pp-caa₃ SU IIc analyzed by ConSurf 2016 [39] revealed three conserved residues, Ala280, Gln283 and Phe293, on a flat region located on the His282 side of the haem c group. Of these residues, only Ala280 is conserved in Tt-caa₃, suggesting possibly different ways of interactions between the SU IIc and its soluble electron donor in T. thermophilus and KF707 species [11,42] (see Discussion).

3.3. A di-haem cyt c₄ is the electron donor to KF707 caa₃-Cox

The KF707 genome contains three putative cyt c₄ (BAU71765, BAU75530 and BAU75507) and two cyt c₅ (BAU77240 and BAU71764) genes (11). Of these, BAU71765 and BAU71764, corresponding to the cyt c₄ and cyt c₅, respectively, are clustered together. The c-type cyt profiles obtained by TMBZ SDS-PAGE gels from membranes of KF707 W.T. and mutant strains grown in glucose or biphenyl, show only one cyt c protein band of 17 kDa (Figure 2 and Table 2). This protein was identified by MS analysis as the di-haem cyt c₄ encoded by the BAU71765 gene (Table 2). In order to confirm this identification and clarify the role of cyt c₄ (BAU71765) or cyt c₅ (BAU71764) as possible electron donor(s) to KF707 caa₃-Cox, three different deletion mutants (KFΔc₄, KFΔc₅ and KFΔc₄/c₅) were constructed (Table 1 and Table S3). The NADI test, which indicates the Cox activity, was mostly negative for KFΔc₄ and KFΔc₄/c₅ mutants (Figure S4), but was positive for KFΔc₅ cells like the W.T. strain, indicating that only the absence of cyt c₄, but not that of c₅, affected KF707 Cox activity. Based on these and other findings (e.g., growth rates of mutants, not shown), the function of cyt c₅ (BAU71764) in the KF707 respiratory chain was not pursued further.

The TMBZ SDS-PAGE analyses of W.T. and KFΔc₄ membranes prepared from cells grown on glucose or biphenyl (Figure 5) indicated that the 17 kDa protein band corresponding to cyt c₄ was absent in KFΔc₄ mutants, while all of the remaining c-type cyts were present. Apparently, the lack of cyt c₄ did not affect the regulation of KF707 terminal oxidases, a finding which was also confirmed using appropriate translational lacZ fusion constructs (not shown). In order to unequivocally establish the role of cyt c₄ as an electron donor to KF707 caa₃-Cox, the triple mutant KFΔc₄/cco1-2 was constructed (Table 1). The rate of oxygen consumption initiated by addition of NADH (or ascorbate/TMPD) as electron donors was determined (Table 3). The data showed that in both KFΔc₄ and KFΔc₄/cco1-2 membranes from cells grown either with glucose- (Glu) or biphenyl- (Bph), NADH respiration was largely insensitive to 50 μM cyanide (CN⁻). This chemical is known to fully inhibit both caa₃- and cbb₃1-2 Cox (11). Thus, in the absence of cyt c₄, most of the NADH/oxygen consumption is sustained via the CIO (cyanide insensitive oxidase) activity. This latter activity was ~ 2–3 folds higher than the equivalent activity found in W.T. membranes, but similar to that seen with KFΔcco1-2 membranes. Most importantly, the ascorbate/TMPD initiated oxygen consumption activity in KFΔc₄ membranes was 70% and 80% less than that in KF707 W.T membranes from cells grown on glucose or biphenyl, respectively, supporting the crucial role of cyt c₄ in mediating TMPD oxidation via the terminal oxidases. Further, considering that the caa₃-Cox was greatly expressed in biphenyl grown cells (Figure 4), the decrease of the ascorbate/TMPD activity seen by comparing membranes from biphenyl grown KFΔc₄/cco1-2 with those from similarly grown KFΔcco1-2, unequivocally demonstrated that cyt c₄ acts as a physiological electron donor to KF707 caa₃-Cox.

Figure 5.

Profiles of the c-type cytochromes detected in membranes of KF707 W.T. and KFΔc₄ mutant strains grown with glucose or biphenyl as the carbon source until stationary phase (OD_{600 nm} 0.8–1.0). Membrane fragments were analyzed by SDS-PAGE, and the c-type cytochromes were revealed via their peroxidase activities using TMBZ staining, as in Figure 2.

Table 3.

Respiratory activities in KF707 membranes from W.T. and appropriate mutant cells grown until the stationary phase (OD_{600 nm} 0.7–0.9) in glucose (Glu) or in biphenyl (Bph) as the sole carbon source. See the text for further details.

	Substrate
Carbon source/Strain	NADH	NADH/CN−	Asc-TMPD	Asc-TMPD/CN−
Glu/KF707 W.T.	158 ± 9,0	31 ± 5,0	150 ± 15	10 ± 2,0
Glu/KFΔc4	67 ± 2,0	55 ± 4,0	52 ± 2,0	4,5 ± 1,0
Glu/KFΔcco1-2	160 ± 8,0	114 ± 0,1	38 ± 1,0	8,0 ± 1,0
Glu/KFΔc4/cco1-2	85 ± 6,0	80 ± 5,0	10 ± 1,0	8,0 ± 1,0
Bph/KF707 W.T.	110 ± 3,0	10 ± 0,5	161 ± 12	8,0 ± 2,0
Bph/KFΔc4	34 ± 2,0	31 ± 2,0	32 ± 2,0	3,0 ± 1,0
Bph/KFΔcco1-2	97 ± 8,0	27 ± 1,5	115 ± 11	5,0 ± 1,5
Bph/KFΔc4/cco1-2	28 ± 1,5	27 ± 1,5	15 ± 1,0	5,3 ± 1,0

Non standard abbreviations used: Asc, sodium ascorbate (5 mM); TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine (50 μM); CN⁻, potassium cyanide (50 μM). Mutant strains: KFΔc₄, lacks cytochrome c₄; KFΔcco1-2, lacks both cbb₃1- and cbb₃2-Cox; KFΔc₄/Δcco1-2, lacks cytochrome c₄ and the cbb₃-1- and cbb₃2-Cox. Oxygen consumption rates are expressed as nmoles of O₂ consumed min⁻¹ mg of protein⁻¹, are the mean values of at least two to three assays.

4. Discussion

Most bacteria possess multiple respiratory terminal oxidases that differ in their proton pumping efficiency and oxygen affinity, enabling energy transduction under various O₂ tensions and with different types and amounts of carbon sources available to cells, as shown for R. sphaeroides, P. aeruginosa PAO1 and Shewanella oneidensis MR-1 [13,15,40,43–47]. In Pseudomonas sp. B4, which is a PCB degrader closely related to KF707, a shift from growth in glucose to a medium with biphenyl was shown to induce a stress response highlighted by accumulation of inorganic polyphosphate and production of the general stress protein GroEL [44]. Although our present knowledge on KF707 cell stress response is quite limited [18,45], the use of biphenyl as a carbon source is likely to generate a stress response, as suggested by prolonged growth phase initial lag (several times longer than that seen in glucose) [11]. In this respect, the RelA/SpoT Homologue (RSH) proteins are known to play key roles in regulating the cellular response to nutrient stress signals [46]. Indeed, the KF707 RelA/SpoT double mutants (KFΔrelA/spot) show a longer initial lag phase as compared with the W.T. cells when grown with aromatic carbon sources like biphenyl or benzoate. Moreover, in these mutants the expression of the terminal oxidases does not respond anymore to the carbon source, unlike the W.T. strain, suggesting that RelA/SpoT proteins may be involved in this regulation as a stress response (unpublished data).

The expression levels of the terminal oxidases affect the respiratory energy transduction and the membrane electron transport rates, which in turn reflect the capacity of soluble redox carriers to connect the terminal oxidases with other membrane-integral redox complexes [8]. As these connections might be rate-limiting, bacterial species have adopted strategies to overcome these restrictions by forming respiratory super-complexes, or fusing a c-type haem electron carrier to a terminal oxidase subunit [8]. In the thermophiles T. thermophilus and R. marinus and the spore-forming B. subtilis the subunit II of an aa₃-Cox has an extra domain carrying a c-type haem [1,48,49]. In S. oneidensis MR-1, a C-terminal extension of the aa₃-Cox subunit II has two putative c-type haem binding sequences [40]. Interestingly, this fusion oxidase (named Ccaa₃) is expressed under nutrient-starved conditions, similar to the aa₃-Cox of PAO1 [15,47]. Analogous c-type haem domains containing aa₃-Cox were also reported in the anaerobe Desulfovibrio vulgaris [50] and in the anoxygenic phototrophs Rubrivivax gelatinosus and R. sphaeroides [51,52]. Thus, the aa₃-Cox with additional c-type haem containing subunits are frequently encountered among the bacterial genera and habitats [8]. The finding that KF707 also contains a caa₃-Cox expressed in cells grown on biphenyl [11] now extends the occurrence of this type of metabolically regulated Cox enzymes to the Pseudomonas species. Here, using TMBZ/SDS-PAGE and Nano LC-MS/MS analyses, we demonstrated for the first time that the 37 kDa SU IIc of KF707 Caa₃-Cox indeed contains a c-type haem. Moreover, this SU IIc is more abundant in membranes from cells grown on biphenyl than those on glucose, in agreement with the previously determined promoter activities of caa₃-Cox, using cox1-lacZ fusion constructs [11]. In light of these findings, the amino acid sequence and predicted structure of KF707 caa₃-Cox (Pp-caa₃) were compared with those from T. thermophilus (Tt-caa₃) (Figure S3). A large part of the haem c pocket of SU IIc, especially on the His282 side, is conserved between the two species. In contrast, the strand-loop-strand motif forming the bottom part of this haem c pocket in Tt-caa₃ (residues 273-284 in T. thermophilus numbering) is not present in Pp-caa₃, where it is replaced by a large insertion (residues 129-153 in Pp-caa₃, numbering Tt-caa₃). As this insertion is missing in Tt-caa₃, this portion of the model built here relied on the structure of P. denitrificans aa₃-Cox (Pd-aa₃), and suggested that the haem c pocket of Pp-caa₃ might be a combination of that from Tt-caa₃ and Pd-aa₃ together.

Comparing the homology model of Pp-caa₃ with the structure of Tt-caa₃ we note that the electron transfer pathway seen between the SU IIc haem c and its homo-binuclear Cu_A center is highly conserved in Pp-caa₃, except for a few conservative substitutions. This observation suggests that the electron transfer pathways from Cu_A to aa₃ haems in both enzymes are similar (Figure 4C). Likewise, the two proton channels (D- and K-pathways) located in SU I of the aa₃-Cox [1], required for chemical reduction of O₂ in the haem-Cu binuclear center, are conserved between the Tt-caa₃ and Pp-caa₃, with most of the residues constituting these pathways being conserved, or conservatively substituted (Figure 6A and B). A major difference is observed at the end of the D-pathway, near haem a_S3, where Tt-caa₃ Gln84, Ser249 and Thr252 were substituted with Pro91, Gly256, and Glu259 in Pp-caa₃. Apparently, the so-called YS gateway seen in the Tt-caa₃ structure [5] may not be present in of Pp-caa₃. Whether these differences constitute the basis for our inability to obtain a viable KF707 mutant containing only the caa₃-Cox, remains unknown [11]. Somewhat similarly, a P. aeruginosa PAO1 mutant that contains only the aa₃-Cox was also unable to grow aerobically [14], unless it contained a suppressor mutation in the two-component regulatory system RoxSR [16]. Work is in progress to see whether a similar situation also occurs in KF707.

Figure 6.

Proton D- and K-pathways (A and B, respectively) in KF707 caa₃-Cox model structure. The D-pathway in the Tt-caa₃ structure begins near to Asp103 and leads through a solvent-filled cavity to Tyr248 in the vicinity of SU I haem a_S3. In the mode, Tt-caa₃ Tyr248 corresponds to Pp-caa₃ Phe255. The K-pathway originates at Tt-caa₃ with Glu84 of SU IIc and continues up to the binuclear copper center Cu_B via the cross-linked SU I/III His250-Tyr254, by means of Lys328. These residues are fully conserved in the Pp-caa₃ model structure shown here (SU IIc Glu78 and SU I His257-Tyr261 and Lys335). The protein backbone is reported as ribbons colored as in Figure 3. The haem groups are in ball and stick, with the iron and copper metal centers as orange and ocher spheres, respectively. The Mg(II) ion in SU I is shown as a green, while the water molecules (W) are depicted as red spheres. The residues mentioned in the text are in sticks colored according to the atom type.

Structural comparisons of Tt-caa₃ and Pp-caa₃ raise intriguing issues, such as the membrane-external surface of Pp-caa₃ SU IIc, which has three residues (Ala280, Gln283 and Phe293) of which only one (Ala280) is conserved in Tt-caa₃. A possibility is that this difference might reflect the presence of different electron donors to the SU IIc of T. thermophilus versus that of KF707 caa₃-Cox. In the case of KF707, this work showed for the first time in a Pseudomonas species that the 17 kDa di-haem c₄ (BAU71765) functions as the main electron donor to the caa₃-Cox (Table 3). In the case of T. thermophilus caa₃-Cox, although the multi-domain di-haem cyt c₅₅₀ acts as an electron donor [53], this subject remains a matter of controversy [42].