The membrane-attached electron carrier cytochrome c_y from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer

Abstract

Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c₂^Rc and a membrane-bound bipartite Cyt c_y^Rc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c₂^Rs. Recent work indicated that this species has a gene, cycY^Rs, that is highly homologous to cycY^Rc, and in the work presented here, functional properties of its gene product (Cyt c_y^Rs) are defined. It was found that Cyt c_y^Rs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt c_y^Rc. Chimeric constructs have shown that the photosynthetic incapability of Cyt c_y^Rs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?

Mitochondria of higher eukaryotes contain a single, soluble, and highly conserved cytochrome (Cyt c) that functions as an electron carrier between the membrane-associated Cyt bc₁ complex and the aa₃-type Cyt c oxidase in the linear electron-transfer chain (ETC) of aerobic respiration. On the other hand, in α-proteobacteria, which are used widely for functional and structural studies on biological energy transduction, several aerobic and anaerobic ETCs are used under different growth conditions. In these bacteria, the presence of multiple ETCs creates a physiological need to funnel electrons specifically to appropriate destinations, depending on the growth conditions. Thus, in species like Rhodobacter capsulatus and Paracoccus denitrificans, unlike mitochondria, multiple Cyt c function in parallel as electron carriers with seemingly overlapping functions (1).

Photosynthetic (Ps) and respiratory (Res) ETCs of the purple, nonsulfur facultative phototrophs of Rhodobacter species provide an excellent model system for studies on cellular energy transduction (2). In these species, Ps growth depends on the cyclic electron transfer between the photochemical reaction center and the Cyt bc₁ complex. In R. capsulatus, either a soluble Cyt c₂, or a membrane bound Cyt c_y, reduces the photooxidized reaction center from the periplasmic side of the cytoplasmic membrane (3). The same electron carriers also transfer electrons from the Cyt bc₁ complex to the cbb₃-type Cyt c oxidase (4), thereby participating in a mitochondrial-like Res ETC of this species (5). However, in wild-type strains of the closely related Rhodobacter sphaeroides, Ps growth strictly depends on the presence of the soluble Cyt c₂ (6). Thus, it was believed that a Cyt c_y homolog is not present in R. sphaeroides. In the absence of any other suitable candidate, participation of Cyt c₂ as an electron carrier from the Cyt bc₁ complex to the aa₃-type (7) and to the cbb₃-type (8) Cyt c oxidase also has been proposed (6). Recently, a gene highly homologous to Rc cycY (encoding Cyt c_y^Rc) was encountered in R. sphaeroides (9). This finding suggested to us that R. sphaeroides Cyt c_y (Cyt c_y^Rs) might not be functional in Ps electron transfer, because a Cyt c₂⁻ R. sphaeroides strain is Ps⁻ (6) and also is complemented readily to a Ps⁺ phenotype with Cyt c_y^Rc (10). Herein, we have identified the polypeptide corresponding to Cyt c_y^Rs and characterized its functional properties. We show that Cyt c_y^Rs is a membrane-attached electron carrier that participates only in Res electron transfer, unlike its R. capsulatus counterpart, which is functional for both Ps and Res electron transport. Furthermore, using chimeric Cyt c_y constructs, we establish that the differential Ps capability of these cytochromes is related, at least partly, to their redox active domains interacting with their partners. It therefore seems that, although the Rhodobacter species simultaneously express several highly homologous electron carriers, they also endow them with pronounced specificity toward their redox partners to direct electron flow efficiently to the different branches of their ETCs, thereby maximally supporting their physiological needs.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions.

R. capsulatus and R. sphaeroides strains were grown under chemoheterotrophic or photoheterotrophic conditions in mineral-peptone-yeast extract (MPYE)-enriched (11) and yeast-extract-casamino acid-enriched (12) media, respectively, or in minimal medium A, as described (11). Photoheterotrophic growth on solid medium occurred in anaerobic jars with H₂/CO₂-generating gas packs (BBL). Res and Ps growth rates were determined as described (13, 14). Escherichia coli strains CJ236 [dut1 ung1 thi-1 relA1/pCJ105 (F′ Cam^R)] (15), HB101 [F⁻proA2 hsdS20 (r_B⁻, m_B⁻) recA13 rpsL20] (15), S17.1 [ara recA rpoL RP₄-2-2-Tet∷Mu-Kan∷Tn7] (16), and XL1-Blue [recA1 endA1 gyrA96 thi-1, hsdR17 lac/F′ proAB lacI^qZΔM15 Tn10 (Tet^R)] (15) were grown on Luria–Bertani broth, supplemented, where appropriate, with antibiotics, as described (11).

Molecular Genetic Techniques.

Uracil-containing single-stranded DNA isolated from E. coli CJ236 containing pKD2 (cycY^Rs) and the primer 5′-CTGGCCTCGCTCCAGGACTACAAGGACGACGATGACAAGTAAGGGCAGAACCCCCTG-3′ was used to extend by site-directed mutagenesis the C terminus of Cyt c_y^Rs with an in-frame octapeptide corresponding to the FLAG epitope (N-Asp–Tyr–Lys–Asp–Asp–Asp–Asp–Lys-C; Kodak) followed by a stop codon, as it has been described for Cyt c_y^Rc (11). This process yielded pHM118 (cycY^Rs-FLAG). For mutagenesis of pHM6 carrying cycY^Rc-FLAG (11), the QuikChange kit (Stratagene) was used as recommended by the manufacturer, with the following modifications. All reaction mixtures contained 15% (vol/vol) glycerol and were incubated at 98°C for 30 s before cycling (18 cycles of 97°C for 30 s, 55°C for 10 s, and 68°C for 12 min). The mutagenic primers NdeI.1 (5′-CGCCGGGGGACATATGCTCGTCAAGACGCAC-3′) and NheI.1 (5′-CGAAGTGGACCCGGCTAGCATCACCGGCGAC-3′) were used to create an NdeI site at base-pair position 87 of the insert (corresponding to methionine 1 of Cyt c_y^Rc; pHM110) and an NheI site at base-pair position 371 (changing Thr-96 of Cyt c_y^Rc to Ser; pHM112), respectively. Plasmid pHM113 was obtained by using pHM110 as a template and had both of the aforementioned NdeI and NheI sites.

DNA Sequence Analysis.

Automated DNA sequencing with a dye terminator cycle sequencing kit (AmpliTaq FS, Applied Biosystems) was performed as specified by the manufacturer. The nucleotide sequence of the 1.25-kb insert on pKD2 was determined on both strands by using the M13 universal primers and the following internal primers obtained through GIBCO/BRL: scycY1, 5′-CGTCACATCCTCGCCGTG-3′; scycY4, 5′-CGCCGAGGCGCCGACCTGGA-3′; scycY2, 5′-TGGAAAGCGATTCGGCCGGT-3′; scycY5, 5′-GGCCGAATCGCTTTCCAGGG-3′; scycY3, 5′-TCGCCAAATGCGCCGCCTGTC-3′; and scycY6, 5′-GGCCACCGCGCGGTCGAC-3′. Homology searches, sequence alignments, and predictions for transmembrane helices were performed as described (11).

Construction of Chimeric Cyt c_y Derivatives.

PCRs were performed by using AmpliTaq DNA polymerase in the presence of 200 ng of each primer and with 20–50 ng of plasmid DNA as a template (11). For the “Cyt c exchange” construct, the Cyt c domain of Cyt c_y^Rs-FLAG from pHM118 was amplified (30 cycles of 97°C for 30 s, 55°C for 10 s, and 72°C for 2 min) by using the universal M13 forward primer and the primer 5′-GGAAGCGAATTCGCCAAATGCGCCGCC-3′ carrying an EcoRI site. The 0.8-kb PCR product thus obtained was digested with XbaI and EcoRI restriction enzymes and used to replace the corresponding fragments of pHM4 containing the “anchor-linker” domain of Cyt c_y^Rc fused in-frame to the mature form of Cyt c₂ (11), yielding pHM124. This plasmid encoded a chimeric Cyt c_y where the first 108 amino acid residues of Cyt c_y^Rc are fused in-frame to the Cyt c domain (residues 82–174) of Cyt c_y^Rs-FLAG. For the anchor-linker exchange construct, residues 1–68 of Cyt c_y^Rs were amplified by using pKD2 as a template and the primers 5′-CGAATCCATATGTTCGACACGATGACCCTG-3′ and 5′-GTAACTGCTAGCTTCCGGAAACGGCACCGCTT-3′. The PCR product was digested with NdeI and NheI and ligated to the 3.9-kb NheI–NdeI fragment of pHM113, yielding pHM121. Because the NdeI mutation overlaps the ATG start codon of cycY^Rc, it affected the expression of Cyt c_y^Rc. Thus, the NdeI mutation in pHM121 was converted back to the wild-type sequence by using the mutagenic primer 5′-AACCGCCGGGGGAGCCATGTTCGACACGATG-3′. The resulting plasmid pHM125 encoded a chimeric Cyt c_y in which the first 68 amino acid residues of Cyt c_y^Rs are fused in-frame to the Cyt c domain (residues 95–199) of Cyt c_y^Rc-FLAG. All constructions were confirmed by restriction digestions and DNA sequencing analyses, and the plasmids pHM124 and pHM125 were cloned into the unique HindIII site of pRK415 (17) to yield their transferable derivatives pHM126 and pHM127, respectively (Table 1).

Table 1.

Bacterial strains and plasmids

Strain	Relevant characteristics	Refs.178
R. capsulatus
MT1131†	crtD121 RifR, wild-type	13
Y262	Overproducer of gene transfer agent	18
FJ2	crtD121 Δ(cycARc∷kan) Δ (cycYRc∷spe); cyt c2Rc−cyRc−,Ps−	3
M6G-G4/S4	crtD121 qox-260 Δ(cycA∷kan); Qox− cyt c2−,Ps+	14
R. sphaeroides
Ga†	crt, wild-type	10
Gadc2	crt, Δ(cycARs∷spe); cyt c2Rs−,Ps−	19
Gadcy	crt, cycYRs∷kan; cyt cyRs−,Ps+
Gadc2cy	crt, cycYRs∷kan Δ (cycARs∷spe); cyt c2Rs−, cyt cyRs−,Ps−
Plasmids
pRK2013	KanR, helper plasmid	17
pRK415	TetR broad host-range plasmid	17
pSUP202	AmpR, CmR, TetR, suicide plasmid	16
pBSII	pBluescript II KS(+), AmpR	11
pFJ66	Δ(cycY∷spe); SpeR, TetR	3
pKD2	cycYRs on a 1.25-kb EcoRI/BlgII fragment of pUI8180 in pBSII
pHM4	cyt MA-c2 on a 1.2-kb BamHI/HindIII fragment in pBSII	11
pHM6	cycYRc-FLAG on a 1.2-kb BamHI/HindIII fragment in pBSII	11
pHM8	cyt MA-c2 on a 1.2-kb KpnI/BamHI fragment in pRK415	11
pHM13	cycYRs on a 2.0-kb XbaI/HindIII fragment in pRK415
pHM14	cycARc on a 1.25-kb BamHI/HindIII fragment in pRK415
pHM15	cycYRs on a 1.6-kb EcoRI/XbaII fragment of pU1942 (see ref. 9) in pBSII
pHM16	cycYRs∷kan on a 2.9-kb EcoRI fragment in pBSII
pHM17	cycYRs∷kan on a 2.9-kb EcoRI fragment in pSUP202
pHM100	Like pHM126 but without a FLAG epitope
pHM110	Like pHM6 but contains an NdeI site at position 87
pHM112	Like pHM6 but contains an NheI site at position 371
pHM113	Like pHM6 but contains both an NdeI and an NheI site at positions 87 and 371, respectively
pHM118	Like pKD2 but contains cycYRs-FLAG
pHM119	pHM118 at HindIII site of pRK415
pHM121	cycYRs68∷cycYRc95-FLAG on a 1.2-kb KpnI/XbaI fragment in pBSII (“anchor-linker exchange chimera”)
pHM123	pHM121 at HindIII site of pRK415
pHM124	cycYRc108∷cycYRs82-FLAG on a 1.2-kb KpnI/XbaI fragment in pBSII (“cyt c exchange chimera”)
pHM125	Like pHM121 but does not contain an NdeI site
pHM126	pHM124 at HindIII site of pRK415
pHM127	pHM125 at HindIII site of pRK415

¹⁷⁸If not otherwise indicated, all strains and plasmids were obtained during this work. 

^†The R. capsulatus strain MT1131 (Rif^R crtD) and R. sphaeroides strain Ga (crt) are referred to as wild-type strains, because they are wild-type in respect to their Cyt c profile and growth properties. 

Chromosomal Inactivation of cycY^Rc and cycY^Rs.

The null allele of cycY^Rc [Δ(cycY^Rc∷spe)] carried by pFJ66 was introduced into the chromosome of appropriate R. capsulatus strains by using the gene transfer agent (18) as described (3). Chromosomal location of the inserted spectinomycin resistance (Spe^R) cartridge was confirmed by PCR, by using the primers 5′-GAAAGCCGGCGAGGAAAAGT-3′ and 5′-CTCGGTTTTCTGGAAGGCGA-3′ as described (11). The 1.6-kb BamHI fragment of pUC4-Kixx (Amersham Pharmacia) carrying the kanamycin resistance (Kan^R) gene was cloned into the unique BamHI site, overlapping with the glycine 24 codon of Cyt c_y^Rs on pHM15 (Table 1). The 2.9-kb EcoRI fragment of pHM16 thus obtained, transcribing kan and cycY^Rs in the opposite orientation, was cloned into the unique EcoRI site of pSUP202 (16), yielding pHM17 (cycY^Rs∷kan). Conjugal transfer of pHM17 from E. coli S17.1 into R. sphaeroides was done as described (19), and Kan^R transconjugates were screened for tetracycline sensitivity (Tet^S). The chromosomal location of kan in the retained Kan^R Tet^S isolates was confirmed by PCR amplification by using the primers scycY1 and scycY2 described above.

Biochemical Techniques.

Isolation of chromatophore vesicles, SDS/10% or 16.5% PAGE, and immunoblot analyses with anti-FLAG antibodies were performed as described (11). Protein concentrations were determined by the method of Lowry et al. (20), and the presence of Cyt c was indicated by the intrinsic peroxidase activity of the Cyt c heme group by using 3,3′,5,5′-tetramethylbenzidine (TMBZ) and H₂0₂ (21). Oxygen uptake measurements with various substrates and inhibitors were performed as described (5).

RESULTS

Physical Organization of the Genes Surrounding cycY^Rs.

The cosmid pUI8180 contains chromosomal DNA from the region 443 kb clockwise from the chosen zero position on the physical map of chromosome I of R. sphaeroides (9). Determination of the DNA sequence of its 1.25–kb EcoRI–BglII fragment on pKD2 (Table 1) identified three ORFs (Fig. 1). The largest one of these ORFs (522 bp), encoding a Cyt c of 17.7 kDa (for the apoprotein), with a conserved heme binding motif (Cys–Ala–Ala–Cys–His) between its residues 85 and 89, had pronounced homology to cycY^Rc. Hereafter, this ORF is called cycY^Rs, and the properties of its product, Cyt c_y^Rs, are described below. Genetic organization and sequence of the ORFs either preceding (pheA encoding chorismate mutase/prephenate dehydratase) or following (appA involved in oligopeptide transport, formerly called ORF2) cycY^Rs are well conserved between R. sphaeroides and R. capsulatus (ref. 11; Fig. 1).

Figure 1.

(A) Physical and genetic maps of the chromosomal regions surrounding R. sphaeroides and R. capsulatus cycY. Only cycY^Rs is physically linked to the ccoNOQP–ccoGHIS/rdxBHIS cluster required for the biosynthesis of the cbb₃-type Cyt c oxidase in both organisms (9, 30). Arrows and “T” refer to transcription direction and termination signal at the 3′ downstream region of cycY, respectively. (B) Sequence comparison of the 5′ upstream regions of cycY from R. capsulatus (RccycY), R. sphaeroides (RscycY), and P. denitrificans (PdcycM). Putative −35 (TTGAA) and −10 (TAg/cAT) regions for a σ⁷⁰-like promoter are indicated. Additional conserved elements of unknown significance (indicated by arrows) and putative ribosome binding sites rich in adenosine and guanine residues in the vicinity of the boxed translational start (ATG) codons also are shown.

Primary Structure of Cyt c_y^Rs.

Alignment of the amino acid sequence of Cyt c_y^Rs with Cyt c_y^Rc and its homologs in other species (22, 23) indicated that members of Cyt c_y family have conserved their bipartite domain structure (Fig. 2). Each member contains a carboxyl-terminal Cyt c subdomain and an amino terminal “linker” subdomain preceded by an unprocessed signal sequence-like stretch anchoring it to the membrane (11). Several Lys residues in the Cyt c subdomain of Cyt c_y^Rs (e.g., Lys-8, -13, -72, and -87, in equine Cyt c numbering) thought to be important for the interactions of Cyt c with its redox partners (24, 25) are well conserved, whereas several others such residues (e.g., Lys-7, -25, -27, -73, -86, and -88) are not. In addition, a single residue deletion (corresponding to position 111 of Cyt c_y^Rc) and a three-residue insertion (Ala–Pro–Thr at positions 127–130 of Cyt c_y^Rs) are noticeable. Moreover, major differences are seen in the linker regions connecting the membrane anchor to the Cyt c subdomain. In particular, the linker portion of Cyt c_y^Rc is considerably longer than those of R. sphaeroides, P. denitrificans, and B. japonicum because of the 15- and 7-residue insertions located between positions 45–60 and 84–91 of Cyt c_y^Rc, respectively (Fig. 2).

Figure 2.

Alignment of the amino acid sequences for horse-heart Cyt c: R. capsulatus (RccycY), R. sphaeroides (RscycY), P. denitrificans (PdcycM), and Bradyrhizobium japonicum (BjcycM) Cyt c_y homologs. Black and gray boxes correspond to identical residues and conserved substitutions, respectively. The Cyt c domains and CXYCH heme binding motifs are indicated by an arrow and asterisks, respectively. Dots represent the key Lys residues known to be important for the interaction of equine Cyt c with its various redox partners (24, 25).

Cyt c_y^Rs Does Not Function in Ps Electron Transfer.

R. sphaeroides strains bearing a null allele of cycY^Rs in the presence and absence of Cyt c₂ were sought to probe the function of Cyt c_y^Rs. The strains Gadc_y (Cyt c_y⁻) and Gadc₂c_y (Cyt c₂⁻ and Cyt c_y⁻), obtained as described in Materials and Methods, were Ps⁺ and Ps⁻ like their isogenic parents Ga (wild type) and Gadc₂ (Cyt c₂⁻), respectively. Thus, consistent with earlier works (6, 19), inactivation of cycY^Rs had no effect on Ps growth of R. sphaeroides either on minimal or enriched growth media. Furthermore, the R. sphaeroides strain pHM13/Gadc₂ and the R. capsulatus strain pHM13/FJ2 (Cyt c₂⁻ and Cyt c_y⁻), carrying a plasmid-borne copy of cycY^Rs, also were Ps⁻. These findings indicated that Cyt c_y^Rs does not participate in Ps electron transfer in either R. sphaeroides or R. capsulatus. Finally, all R. sphaeroides strains used here were capable of wild-type-like Res growth (doubling times between 144 and 162 min; data not shown). However, the presence of a branched Res pathway, which depended on a poorly characterized quinol oxidase (Q_ox; refs. 26 and 27), precluded us from assessing the role of the Cyt c₂ and Cyt c_y on the Res ETC of R. sphaeroides.

Identification of Cyt c_y^Rs and Its Expression Under Res and Ps Growth Conditions.

To identify the gene product of cycY^Rs readily, its epitope-tagged derivative (Cyt c_y^Rs-FLAG) was constructed. TMBZ-SDS/PAGE and immunoblot analyses of various R. sphaeroides strains producing Cyt c_y^Rs-FLAG indicated that, among the membrane-associated TMBZ stained bands, only a band of 24 kDa was shifted to a molecular mass of 25 kDa and recognized with anti-FLAG antibodies (Fig. 3). Further, this 24-kDa band was absent in Gadc_y (cycY^Rs∷kan), lacking Cyt c_y^Rs. These findings identified the 24-kDa and 25-kDa bands as native Cyt c_y^Rs and its FLAG-epitope-tagged derivative, respectively (Fig. 3 A and B). They also showed that Cyt c_y^Rs is expressed under both Ps and Res growth conditions in R. sphaeroides, although its steady-state level was lower under Ps growth conditions (Fig. 3C). Moreover, the data also indicated that under Ps growth conditions the 30-kDa and 28-kDa bands, proposed to correspond to the Cyt c_p (with a calculated molecular mass of 31 kDa) and Cyt c_o (27 kDa) subunits of the cbb₃-type Cyt c oxidase (10), were expressed at lower levels in comparison to aerobic growth conditions (data not shown).

Figure 3.

(A) TMBZ-SDS/PAGE analysis of Cyt c of various R. sphaeroides strains. Chromatophore membranes were prepared from cells grown on yeast-extract-casamino acid-enriched medium by respiration, and ≈75 μg of proteins were loaded per lane. Lane 1, Ga (wild type); lane 2, Gadc_y (lacking Cyt c_y^Rs); and lane 3, pHM119/Gadc_y (containing Cyt c_y^Rs-FLAG). The addition of the FLAG epitope changes the molecular mass of Cyt c_y^Rs by ≈1 kDa. (B) Immunoblot analysis of a similar gel with anti-FLAG M2 antibody. Lanes 1 and 2 correspond to pHM119/Gadc_y (Cyt c_y^Rs-FLAG) and Gadc_y, respectively, and contained 25 μg of membrane proteins prepared from cells grown as for A. (C) Immunoblot analysis with anti-FLAG M2 antibody as for B, except that chromatophore membranes (25 μg of total proteins per lane) were obtained from cells of pHM119/Gadc_y (Cyt c_y^Rs-FLAG) grown in yeast-extract-casamino acid-enriched medium in the absence (lane 1) and presence (lane 2) of oxygen.

Cyt c_y^Rs Can Support Res Growth, but Not Ps Growth, in R. capsulatus.

Res growth of R. capsulatus becomes restricted to the Cyt bc₁ → Cyt c oxidase branch solely (hence, growth is sensitive to Cyt bc₁ inhibitors like myxothiazol) when the Q_ox-dependent branch is eliminated by a mutation (14, 28). Simultaneous inactivation of Cyt c₂ and Cyt c_y in such a Q_ox⁻ background is fatal for cells (5), strongly implying that these cytochromes are the sole electron carriers capable of connecting the Cyt bc₁ complex to the Cyt c oxidase. Thus, various Q_ox⁻ R. capsulatus strains carrying either the Cyt c_y^Rc or the Cyt c_y^Rs as the sole electron carrier were constructed to assess their electron-transfer capabilities. Growth properties of these strains showed that, although the Cyt c_y^Rc, Cyt c₂, and its membrane-bound derivative, Cyt MA-c₂ (11), were able to support both Ps and Res growth of R. capsulatus, Cyt c_y^Rs was functional only during Res growth (Table 2). The Res electron carrier ability of Cyt c_y^Rs was confirmed further by direct O₂ uptake (Table 3), as well as NADH-induced cyanide-sensitive Cyt c reduction measurements (data not shown). It was noticed that the endogenous O₂-uptake activity of pHM13/SL3 (Cyt c_y^Rs) was relatively low, but it could reach a level similar to that seen with M6G-G4/S4 (Cyt c_y^Rc) on addition of exogenous Cyt c (Table 3). This finding correlated well the low amount of Res electron-transfer activity found in pHM13/SL3 with the low amount of Cyt c_y^Rs present in pHM119/SL3 (Fig. 4B).

Table 2.

R. sphaeroides Cyt c_y is able to support electron transfer from the Cyt bc₁ complex to the Cyt cbb₃ oxidase in R. capsulatus

Strain	Electron carrier (Cyt c2 or Cyt cy)	Ps	Res, min	Myx178
MT1131	Cyt c2Rc  +  Cyt cyRc	+†	+, 142	+
pRK415/M6G-G4/S4	Cyt cyRc	+	+, 199	−
pHM13/SL3‡	Cyt cyRs	−†	+, 344	−
pHM14/SL3	Cyt c2Rc	+	+, 265	−
pHM8/SL3	Cyt MA-c2Rc	+	+, 264	−
pHM100/SL3§	Cyt c exchange chimera	−	+, 321	−

¹⁷⁸Myx corresponds to respiratory growth in the presence of 10 μM myxothiazol in MPYE medium. 

^†MT1131 has a doubling time of approximately 120 min and forms colonies 1.7–1.9 mm in diameter after 2 days of incubation on MPYE medium under the growth conditions used. “+” and “−” indicate Ps growth similar to that of MT1131 on solid medium and no visible colony formation after 5 days of incubation under the same conditions, respectively. Growth rates were not redetermined in SL3 (Q_ox⁻) background, because, in the more appropriate R. capsulatus strain FJ2 (Cyt c₂⁻ and Cyt c_y⁻), plasmid-borne Cyt c_y^Rc, Cyt c₂^Rc, and its membrane-bound derivative (Cyt MA-c₂^Rc) support Ps growth in MPYE medium with doubling times of 205, 137, and 154 min, respectively, whereas Cyt c_y^Rs (pHM13) and cyt c exchange chimera (pHM100) do not confer any appreciable Ps growth. 

^‡SL3 was obtained by introducing Δ(cycY∷spe) allele into the R. capsulatus strain M6G-G4/S4 (Q_ox⁻ Cyt c₂⁻) carrying the desired electron carrier on a plasmid, as described in Materials and Methods. 

^§No Spe^R derivative of pHM127/M6G-G4/S4 carrying the linker-anchor exchange chimera was obtained on introduction of cycY∷spe under Res or Ps growth conditions, unlike pHM100/M6G-G4/S4 (or pHM126/M6G-G4/S4; not shown) carrying the Cyt c exchange chimera. 

Table 3.

Respiratory electron transport activities of various R. capsulatus strains measured by using isolated membrane fragments

Substrates, concentration	Moles of O2 consumed per h per mg of protein
	pRK415/M6G-G4/S4 (Cyt cyRc)	pHM13/SL3 (Cyt cyRs)	pHM8/SL3 (Cyt MA-c2Rc)
NADH, 0.5 mM	9.6	2.2	15.2
Rotenone, 5 μM	0.4	0.4	0.3
Myxothiazol, 2 μM	0.4	0.3	0.2
Antimycin A, 2 μM	1.7	0.5	1.8
Cyanide, 50 μM	0.2	0.1	0.2
NADH + Cyt c, 50 μM	19.2	19.0	21.4

Numbers are the mean value of two sets of independent experiments. Cyt MA-c₂^Rc, membrane-bound derivative of Cyt c₂. 

Figure 4.

(A) Schematic representation of the chimeric Cyt c exchange (pHM124/126) and linker-anchor exchange (pHM125/127) cytochromes along the native Cyt c_y^Rs and Cyt c_y^Rc. Control region, Anchor/Linker, and Cytochrome c refer to the various subdomains of these cytochromes. The restriction sites used to create these chimeras are indicated. (B) Immunoblot analysis of various R. capsulatus strains carrying chimeric Cyt c_y-FLAG derivatives with anti-FLAG M2 antibody. Chromatophores were prepared from cells grown in enriched MPYE medium under either Ps (lanes 1–3) or Res (lanes 4–6) growth conditions, and 25 μg membrane proteins were loaded per lane. Lane 1, pHM126/M6G-G4/S4 (Cyt c exchange); lane 2 and 5, pHM127/M6G-G4/S4 (linker-anchor exchange); lane 3, pHM119/M6G-G4/S4 (Cyt c_y^Rs-FLAG); lane 4, pHM126/SL3 (Cyt c exchange); and lane 6, pHM119/SL3 (Cyt c_y^Rs-FLAG).

Why Is Cyt c_y^Rs Unable to Support Ps Electron Transfer?

Chimeric Cyt c_y derivatives were constructed to determine why Cyt c_y^Rs is unable to function as an electron carrier between the reaction center and the Cyt bc₁ complex (Fig. 4A). First, the Cyt c domain from Cyt c_y^Rs-FLAG was fused to the anchor-linker domain of Cyt c_y^Rc, yielding a Cyt c exchange chimera carried by pHM126. Second, the anchor-linker domain of Cyt c_y^Rs was fused to the Cyt c domain of Cyt c_y^Rc-FLAG, yielding an anchor-linker exchange chimera carried by pHM127 (Table 1). In all cases, caution was taken to reassure that expression of the chimeric cytochromes was mediated by an intact cycY^Rc control region known to be active under both Ps and Res growth conditions in both R. capsulatus and R. sphaeroides (3, 11). Interestingly, the Cyt c exchange chimera encoded by pHM126 was unable to support as the sole electron carrier the Ps but able to support the Res growth of a Q_ox⁻ mutant of R. capsulatus lacking both Cyt c₂ and Cyt c_y (Table 2). However, no derivative of M6G-G4/S4 (Q_ox⁻) lacking both the Cyt c₂ and Cyt c_y but carrying the linker-anchor exchange chimera encoded by pHM127 was obtained (Table 2), indicating that such a strain was unable to grow by either Ps or Res growth. Immunoblot analyses of appropriate strains indicated that pHM126/SL3 readily produced the Cyt c exchange chimera under both Ps and Res conditions. On the other hand, no linker exchange chimera was detectable in strains harboring pHM127, including M6G-G4/S4 (Fig. 4B). Data similar to those presented here (Table 2 and Fig. 4) also were obtained with similar chimeric cytochrome constructs with different fusion junctions (data not shown). The findings, therefore, indicate that the Ps-growth inability of Cyt c_y^Rs seems to be an intrinsic property of its Cyt c domain.

DISCUSSION

This work identified a heme-stainable polypeptide with a molecular mass of 24 kDa as the mature form of Cyt c_y^Rs encoded by cycY^Rs as the R. sphaeroides homolog of R. capsulatus Cyt c_y^Rc. The DNA sequences adjacent to the translational start and stop sites of cycY homologs from several species indicated that the homology was not limited only to their coding portions (Fig. 1). The presence of conserved sequences similar to the −35 and −10 regions and closely resembling consensus promoter sequences recognized by the housekeeping RNA polymerase (29) at the 5′ upstream and the presence of a strong transcription terminator sequence at the 3′ end downstream of cycY suggest that these genes form monocistronic units. It is noteworthy that only in R. sphaeroides is the pheA-appABE cluster containing cycY physically linked to hemZ (encoding an anaerobic coproporphyrinogen III oxidase), fnrL (oxygen-response regulator), and ccoNOQP (Cyt cbb₃ oxidase) genes (9, 30). In R. capsulatus, these genes are dispersed into three different chromosomal loci (31).

The finding that Cyt c_y^Rs does not play a significant role in Ps growth of R. sphaeroides also is consistent with earlier studies performed with mutants lacking Cyt c₂^Rs (6, 10). Indeed, these mutants do not have any appreciable electron-transfer activity to the reaction center even though they harbor Cyt c_y^Rs (10). Considering that Cyt c_y homologs participate in Res electron transfer in some other species (22, 23) and that an unknown membrane-bound Cyt c has been proposed as an electron donor to the aa₃-type Cyt c oxidase in R. sphaeroides (32), we studied whether Cyt c_y^Rs mediates electron transfer to the Cyt c oxidase(s). Growth phenotypes and biochemical assays of appropriate strains showed that Cyt c_y^Rs is able to replace R. capsulatus endogenous Cyt c_y and Cyt c₂ only for Res electron transfer. In addition, its Cyt c domain was sufficient for this activity as indicated by the Cyt c exchange chimera encoded by pHM126 (Table 1). Thus, the reason why Cyt c_y^Rs, which is able to donate electrons efficiently to the Cyt c oxidase, is unable to do so to the reaction center is intriguing. It is noteworthy that only some of the Lys residues of equine Cyt c thought to be important for interactions with its redox partners are conserved in Cyt c_y^Rs (Fig. 2). In addition, the Cyt c₂ binding surface of the reaction center is negatively charged (33, 34), and there is a marked difference between the calculated isoelectric points of Cyt c_y^Rs (pI = 4.74) and Cyt c_y^Rc (pI = 7.76). It may be that the precise location of the charged residues affects the ability of Cyt c_y^Rs to form a productive complex with the reaction center under physiological conditions. This has been proposed to be the case for acidic isocytochrome c₂ (pI = 4.87) of R. sphaeroides, which has ≈40-fold lower affinity for the reaction center in vitro compared with Cyt c₂^Rs (pI = 7.01). It is thought that the negative charge of isocytochrome c₂ brings about its lowered affinity toward the reaction center by changing the orientation of the dipole moment at the isocytochrome c₂ docking surface (35). In any event, a relatively low affinity for binding to the reaction center of the Cyt c domain of the Cyt c_y^Rs may explain, at least partially, the Ps incapability of Cyt c_y^Rs and suggests that its interactions with the Cyt c oxidase may be different than those with the reaction center. In this regard, it remains to be seen whether Cyt c_y^Rs is a preferred electron carrier either for the aa₃-type or the cbb₃-type Cyt c oxidase in R. sphaeroides.

It is interesting to note that the linker-anchor exchange chimera encoded by pHM127 is not detected in R. capsulatus cells grown by either photosynthesis or respiration. Because the transcriptional and translational regulatory regions of this construct are identical to those of cycY^Rc, which is known to be active under both growth conditions (36), it is likely that the absence of this chimera is caused by posttranslational events. If so, it seems that the structure of the linker regions of various Cyt c_y homologs plays an important role in determining their relative steady-state stability and also specificity, possibly by mediating appropriate interactions with their redox partners (22, 23, 36).

Finally, it is widely believed that the ancestor of the mitochondria was a proteobacterium, similar to those investigated herein, carrying both the Cyt c₂ and Cyt c_y (37). Interestingly, a homolog of Cyt c_y^Rs, but not of Cyt c₂, has been uncovered recently in the genome of Rickettsia prowazekii (38) that is related closely to mitochondria, as determined by phylogenetic analyses. However, no homolog of Cyt c_y has been detected in mitochondria, which suggests that soluble Cyt c have been favored during the evolution of eukaryotic cells. Excitingly, in addition to the electron-transfer role, Cyt c recently has been implicated as an activator of apoptotic proteases and nucleases (reviewed in ref. 39). This unexpected function of Cyt c requires its diffusion out of the mitochondria, a process that is not readily feasible for membrane-attached Cyt c_y. Thus, the dual function of eukaryotic Cyt c implies why mitochondria might have conserved soluble electron carriers over their membrane-attached relatives.

The work described here shows that, although Cyt c_y^Rc can support both Ps and Res growth of R. capsulatus, its R. sphaeroides homolog, Cyt c_y^Rs, can participate only in the Res electron transfer, at least partly because of the intrinsic properties of its Cyt c subdomain. Therefore, it seems that the different functional properties of various electron carriers clearly endow facultative phototrophs with an excellent way to regulate the flux of electrons through the respiratory and Ps branches of their ETCs.

ABBREVIATIONS

ETC

electron-transfer chain

Ps

photosynthetic

Res

respiratory

Cyt c

cytochrome c

Q_ox

quinol oxidase

TMBZ

3,3′,5,5′-tetramethylbenzidine

MPYE

mineral-peptone-yeast extract