A heterodimeric cytochrome c complex with a very low redox potential from an anaerobic ammonium-oxidizing enrichment culture


  • Editor: Jörg Simon

Correspondence: Takao Fujii, Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Tel.: +81 96 326 3948; fax: +81 96 323 1331; e-mail: fujii@life.sojo-u.ac.jp


A dimeric cytochrome c with an apparent molecular mass of 25 kDa was isolated from an anammox bacterium, strain KSU-1, in a relatively large quantity. This protein was named the NaxLS complex. The spectrum of the oxidized form exhibited a peculiar Soret peak at 419 nm. The reduction of NaxLS was not complete even with the addition of excess dithionite, but was complete with titanium (III) citrate, indicating that the NaxLS complex has a very low redox potential. The genes encoding the two subunits, naxL and naxS, are adjacent on the genome. The deduced amino-acid sequences of the genes showed high identities with those of two genes encoding ‘unknown proteins’ in the genome of Candidatus Kuenenia stuttgartiensis, but had lower identities with other c-type heme proteins. The electron paramagnetic resonance spectra of NaxLS exhibited low-spin signals of two heme species in the range between g=2.6 and g=1.8, which strongly suggested an unusual His/Cys coordination. This unique coordination might account for the low redox potential of the hemes in NaxLS. NaxLS might participate in the transfer of low redox potential electrons in the intracellular anammoxosome compartment or the cytoplasm.


Anaerobic ammonium oxidation (anammox) was discovered in 1995 in a reactor for denitrification in the Netherlands (Mulder et al., 1995). Shortly after, it was reported that anammox is performed under anoxic conditions by novel autotrophic bacteria (Strous et al., 1999). The first anammox bacterium discovered was provisionally named Candidatus Brocadia anammoxidans (Kuenen & Jetten, 2001). Although the bacteria have not been isolated, many kinds of 16S rRNA genes of phylogenetically related anammox bacteria have been registered in nucleotide sequence databases to date.

The genome of the anammox bacterium, Candidatus Kuenenia stuttgartiensis, was investigated and the hypothetical mechanism of anammox was reported based on the annotation of the identified genes and previous biochemical research (Strous et al., 2006). It is found that the genome codes for the large number of c-type cytochrome genes. Redundancy of the genes is regarded as being due to versatility in the energy metabolism of anammox bacteria such as iron and manganese respiration, and anammox reaction (Strous et al., 2006). The expression of some of them would be expected for anammox reaction.

We succeeded in enriching an anammox bacterium in a continuous-flow reactor with a nonwoven polyester biomass carrier (Fujii et al., 2000; Furukawa et al., 2002). A dominant bacterium in the reactor, named strain KSU-1, with a 16S rRNA gene sequence 92.2% identical to that of C. Brocadia anammoxidans, was identified. Thereafter, two multi-c-type heme proteins, hydroxylamine oxidoreductase (HAO) and hydrazine-oxidizing enzyme (HZO), were purified from strain KSU-1 (Shimamura et al., 2007, 2008). In the purification processes of the proteins, we noticed that many kinds of c-type heme proteins besides HAO and HZO were present in the cell of anammox bacterium.

We have focused on the isolation of cytochrome c with a low molecular weight being specific for anammox bacteria. A heterodimeric c-type heme protein with a molecular mass of c. 25 kDa was isolated and was found to have unique features. The isolation and characterization of the novel heterodimeric c-type heme, named the NaxLS complex, are reported in this study.

Materials and methods

Preparation of cell-free extract and purification of the NaxLS complex

The sludge from the culture containing an abundance of strain KSU-1 (>70%) was prepared as described previously (Fujii et al., 2000). The sludge (wet weight: ∼50 g) was suspended in 100 mM Tris-HCl buffer, pH 8.0, containing 20% w/v glycerol, 1 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and subsequently disrupted by sonication and a Teflon homogenizer. Cell debris and membrane fractions were removed by successive centrifugations of 15 000 g for 15 min and 160 000 g for 1 h at 4 °C. To the resulting supernatant (cell-free extract), ammonium sulfate was added to 40% saturation, and the solution was subjected to centrifugation at 15 000 g for 15 min to remove the precipitate. A gel (Toyopearl Butyl-650M) was packed in a column (gel volume, φ1.9 × 15 cm), and equilibrated with 50 mM Tris-HCl buffer, pH 8.0, containing 20% w/v glycerol, 1 mM EDTA and 0.5 mM PMSF, containing ammonium sulfate to 40% saturation. The supernatant was applied to the column, which was then washed with the same buffer containing 10% glycerol. A linear gradient of a decreasing concentration of ammonium sulfate in buffer was used to elute cytochromes. The first eluted peak was collected and successively applied to a gel filtration column (2.0 × 60 cm) packed with a Superdex 75pg gel equilibrated with 20 mM potassium phosphate buffer, pH 8.0, containing 0.2 M potassium chloride. The protein was eluted with the same buffer. The concentration of heme protein in each fraction was always monitored by measuring the A419 nm and A408 nm.

Measurement of spectra of the NaxLS complex

The absolute spectra of the purified NaxLS complex were recorded at 25 °C using a UV/visible spectrophotometer (MPS-2400, Shimadzu, Japan) against the same buffer used for the gel-filtration column chromatography. The wavelength of the spectrophotometer was calibrated to within 0.2 nm using the emission lines of a deuterium lamp at 486.0 and 656.1 nm. The solution of the complex was appropriately diluted and placed into a cuvette, which was capped with a butyl rubber septum. Then, the solution was blown with argon gas through a syringe needle to purge oxygen for more than 5 min. To reduce the protein, a solution containing an appropriate amount of dithionite or titanium (Ti) (III) citrate was added and then the spectrum was recorded.

Measurement of the electron paramagnetic resonance (EPR) spectrum

The NaxLS complex was concentrated to about 0.5 mgprotein mL−1 and the solution buffer was exchanged to 10 mM HEPES buffer, pH 7.0, with an Amicon concentrator. An aliquot of the concentrated sample was kept ice-cold for about 3 h after the addition of excess dithionite. The other was kept at the same temperature for the same period. Each sample was placed in an EPR tube and frozen in liquid nitrogen (77 K). EPR spectra were measured on a Bruker, E-500 spectrometer (X band) with a high-Q resonator and equipped with a variable temperature cryostat (ESR900, Oxford Instruments, UK).

Results and discussion

Purification of the NaxLS complex from an anaerobic ammonium-oxidizing enrichment culture

Column chromatography was used to purify a cytochrome with a low molecular weight. Table 1 shows a summary of the purification of the NaxLS complex in a typical purification procedure. The purified cytochrome was analyzed using HPLC (BioLogic DuoFlow System, BioRad Co., CA) equipped with a gel filtration column (HiLoad 16/60 Superdex 75pg, Amersham Biosciences Co., NJ). The elution profile showed a single peak of protein with an apparent molecular mass of c. 25 kDa (Fig. 1a). The cytochrome was then subjected to Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibiting two bands with molecular masses of c. 14 and 11 kDa on the gel (Fig. 1b). Thus, the protein was likely to be heterodimeric, and was named the NaxLS complex, composed of NaxL and NaxS subunits. Three point 5 mg of the purified protein were obtained from 270 mg of protein in the cell-free extract, indicating about 1.3% w/w recovery from the total protein (Table 1). However, the content must be more than the calculated value of 1.3% (protein recovery) because a significant amount of NaxLS was probably lost in the process of the purification process. Taking into account the high molecular masses of HZO and HAO (c. 130 and 110 kDa, respectively), the molar content of the NaxLS complex in the cell-free extracts is estimated to be comparable to those of HZO and HAO (weight content of c. 10% each).

Table 1.   Summary of the purification of a 25 kDa cytochrome
 Total protein (mg)Protein recovery (%)Heme content (A419 nm/A280 nm)Apparent fold
  1. The purified protein had a specific Soret band at 419 nm.

Cell-free extract2701000.261.0
Supernatant of 40% ammonium sulfate130480.291.1
Toyopearl Butyl-650M eluate5.
Superdex eluate3.51.33.112
Figure 1.

 Estimation of the molecular mass of NaxLS by gel filtration chromatography and Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (a) Elution profile of NaxLS by gel filtration chromatography: the purified protein (500 μL, 1.5 mg mL) was charged on the column, and eluted with 20 mM potassium phosphate buffer, pH 8.0 containing 0.2 M KCl (flow rate of 1 mL min−1). A280 nm of the eluted solution was continuously measured. The inset shows the correlation of the molecular masses of marker molecules with elution volumes. A mixture of five kinds of authentic proteins, conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), RNaseA (13.7 kDa), aprotinin (6.5 kDa), was used as molecular mass markers. (b) A gel image in Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of NaxLS: the purified protein was subjected on 10% Tricine–SDS-PAGE, and stained with Coomassie Brilliant Blue G250. Lane 1, polypeptide standard (BioRad Co.) was applied as a molecular marker; lane 2, 5 μg of the purified protein was applied.

The genes encoding the subunits, NaxL and NaxS, of the protein complex

The nucleotide sequence of a DNA fragment (c. 3 kb) harboring four ORFs, tentatively named ORF I, II, III and IV, was determined (Supporting Information, Appendix S1). ORF I encoded NaxL and ORF II encoded NaxS. ORF II encoded a polypeptide composed of 126 residues. The N-terminus of NaxS started with the 27th residue of the polypeptide, suggesting the presence of a signal sequence of 26 residues. Mature NaxS was composed of 100 residues with a molecular weight of 10 825, and it contained a heme-binding motif specific to c-type heme proteins, CYYCH, between the 28th and the 32nd residues from the N-terminus. On the other hand, ORF I encoded a polypeptide of 110 residues and a preceding signal sequence of 28 residues as predicted by signalp software. Mature NaxL was estimated to have a molecular weight of 12 547. A heme-binding motif, CRNCH, was located between the 16th and the 12th residue from the C-terminus, which is typical of heme proteins belonging to the class II cytochrome c family. Homology searches were performed using the blast program. The deduced amino-acid sequences encoded by naxL and naxS demonstrated the highest identities (60% and 78%) with those of unknown proteins in the genome of C. Kuenenia stuttgartiensis, registered as CAJ70832 and CAJ70833, respectively. The orthologous genes of C. Kuenenia stuttgartiensis also flank each other on the genome (Strous et al., 2006). These results demonstrated that the primary structures of the NaxLS complex and the synteny of the genes are conserved in both anammox bacteria. In contrast, the deduced amino-acid sequence around the heme-binding motif of NaxL exhibited lower identities (∼40%) to those of the corresponding region of a cytochrome c′ (YP_425133) belonging to the class II cytochrome c family. The sequence of NaxS had lower identities to those of class I cytochromes c including cytochrome c552 of C. Kuenenia stuttgartiensis (35%) (AAY86372). The NaxLS complex may be the first cytochrome c composed of class I and class II c-type heme protein subunits.

Absorption spectra of the NaxLS complex

Alkaline pyridine ferrohemochrome of the NaxLS complex prepared according to the previous report (Berry & Trumpower, 1987) showed a typical spectrum for a c-type heme (data not shown). The air-oxidized spectrum of the NaxLS complex showed absorption peaks at 419 and 350 nm, a broad peak at approximately 540 nm and a shoulder at around 580 nm. Upon addition of the reducing reagent dithionite to the oxidized form of the NaxLS complex, the Soret peak moved slowly to the lower wavelength (blue direction) (417 nm) and was only slightly taller for about 15 min at 25 °C with the emergence of small peaks at 547 nm (α-band), 522 nm (β-band) and a shoulder at around 580 nm (Fig. 2a). These spectra indicate that dithionite incompletely reduced the NaxLS complex. In contrast, addition of Ti (III) citrate resulted in the immediate appearance of a Soret peak at 416 nm with relatively large peaks at 553 nm (α-band) and 523 nm (β-band) (Fig. 2b). The spectrum is typical of the reduced form of c-type heme proteins. Because the standard redox potentials of dithionite and Ti (III) citrate at pH 7 are known to be about −400 mV and −800 mV, respectively (Mayhew, 1978; Reijerse et al., 2007), the redox potential of the complex is estimated to be −400 mV or less.

Figure 2.

 UV/visible absorption spectra of purified NaxLS. Insets show expanded spectra in the range between 470 and 620 nm. The concentration of NaxLS was about 60 μg mL−1. Solid and dotted lines represent spectra of NaxLS treated with air and reducing reagents, respectively. Dithionite (a) and Ti (III) citrate (b) were used as reducing reagents.

The absorption peaks of the oxidized form of NaxLS were red-shifted as compared with those of ordinary c-type heme proteins. A similar spectrum is reported in a cytochrome c mutant, Cyt-Cys80, whose native ligand of Met is substituted with Cys to form His/Cys coordination. This mutant exhibits absorption peaks at 416 nm (Soret band) and 540 nm (β-band) (Raphael & Gray, 1991). A nitrogenous substance, such as imidazole and 1-methylimidazole, occupies sixth coordination position of a b-type heme of cytochromes P450 and induces a specific spectrum exhibiting absorption peaks at 419–426 nm (Soret band) and 570 nm (α-band) as a shoulder on the broad β-band at 538–541 nm (Dawson et al., 1982; White & Coon, 1982). Despite the difference in c-type and b-type heme, His/Cys coordination might produce similar spectra. Upon reduction of NaxLS, the spectrum was the usual one as shown to be the case for Cyt-Cys80 (Raphael & Gray, 1991), implying that the thiolate–iron bonds in the ferrous form are no longer intact.

EPR spectra of the NaxLS complex

The EPR spectra of the oxidized form (ferric heme) of NaxLS illustrated two sets of low-spin signals in the range of g=2.6–1.8, indicating the existence of two kinds of low-spin hemes (Fig. 3a). The LS species with g values of 2.522, 2.270 and 1.868 is tentatively denoted as LS1 and the other species with g values of 2.430, 2.250 and 1.910 as LS2. No signals of ferric high-spin heme were observed.

Figure 3.

 EPR spectra of NaxLS in the absence (a) and presence of dithionite (b). The concentration of NaxLS was 0.47 mg mL−1. Experimental conditions were, microwave frequency, 9.3 GHz; microwave power, 1 mW; field modulation amplitude, 10 G; modulation frequency, 100 kHz; averaged scans, five times; temperature, 10 K. Asterisks show g-values of LS1. The values of LS2 are shown without asterisk.

When dithionite was added to the protein solution, the EPR signals of LS1 and LS2 decreased in intensity but the decrease, which was larger in LS1 than in LS2, was <30% even after several hours (Fig. 3b). This indicates that both the low-spin hemes in NaxLS, LS1 and LS2, are quite resistant to the dithionite reduction and this is consistent with the optical results that showed only a small shift of the Soret-band maxima upon reduction with dithionite (Fig. 2a). Only the broad signal at g=2.09 completely disappeared by the reduction. Reduction with dithionite also shifted the g-values of LS1 drastically to 2.570, 2.260 and 1.844 (LS1′). At the same time, the line width of the LS1 signals broadened with an apparent decrease in the intensity. In contrast, the LS2 signals were only slightly affected by reduction and showed very small g-shifts (Fig. 3).

Axial ligand structure of hemes in NaxLS and the related redox nature

The two LS species found in NaxLS are attributable to each redox center of NaxL and NaxS. The EPR results suggest that the two heme sites have not only different redox potentials but also different protein milieus: one (LS1) is flexible and the other (LS2) is fixed. Because the broad signal at g=2.09 is indicative of some spin–spin interaction, the g-shifts of LS1 might be caused, in part, by the decoupling of such an interaction. To assign LS1/LS2 to NaxL/NaxS, the independent expression of each gene in Escherichia coli cell is under way.

The g-values of LS1 and LS2 of NaxLS are remarkably similar to those of the LS species of SoxA of the SoxAX complex, involved in sulfur oxidation of Paracoccus pantotrophus (Cheesman et al., 2001; Reijerse et al., 2007): LS1, g=2.54, 2.30, 1.87 and LS2, g=2.43, 2.26, 1.90. The axial heme ligands of the SoxA LS1 and LS2 are determined to be His-Cys and His-Cys-S, respectively, by X-ray crystallography. Then, the axial heme ligands of LS1 and LS2 of NaxLS are strongly suggested to be His-thiolate. An alignment of the amino acid sequences of NaxS and four other proteins having c-type heme was performed using ClustalW program (Fig. 4). The amino-acid sequences of NaxS and four related heme c proteins (obtained by clustalw program). The heme c of Arthrospira cytochrome c6 has Met/His coordination [Kerfeld et al., 2002; PDB ID, 1KIB; (5) in Fig. 4] and the axial Met is conserved in two other proteins, a heme protein (EES51901) from Leptospirillum [(3) in Fig. 4] and cytochrome c552 (AAY86372) from C. Kuenenia stuttgartiensis [(4) in Fig. 4]. On the other hand, in NaxS [(1) in Fig. 4] and a deduced protein (CAJ70833) from C. Kuenenia stuttgartiensis [(2) in Fig. 4], Cys occupies the Met position.

Figure 4.

 An alignment of the amino-acid sequences of NaxS and homologous proteins. (1) Amino-acid sequence of NaxS from strain KSU-1; (2) the sequence of a deduced protein (CAJ70833) from Candidatus Kuenenia stuttgartiensis; (3) the sequence of a heme protein (EES51901) from Leptospirillum; (4) the sequence of cytochrome c552 (AAY86372) from C. Kuenenia stuttgartiensis; (5) the sequence of cytochrome c6 (P00118) from Arthrospira. Conserved residues are indicated with asterisks and dots. Heme-c-binding motifs (CXXCH) are boxed. Bold characters, H and M, are the ligands identified on the structure of the cytochrome c (PDB ID, 1KIB). M or C residues that are candidates for ligands are boxed with a broken line.

Similar g-values to those of LS1 and LS2 of the NaxLS complex are generally obtained for the b-type hemes with the Cys axial ligand: for example CO-sensor CooA (Cys-Pro, Aono et al., 1998; Lanzilotta et al., 2000), cystathionine α-synthase (His-Cys, Ojha et al., 2000), cytochrome P450cam (Cys-H2O, Dawson et al., 1982) and NO synthase (Dawson et al., 1982; Tsai et al., 1996). In contrast, most cytochromes c participating in electron transfer have His/Met or His/His coordination (Wilks, 2002). The His/Cys coordination in heme c is known to be limited: the aforementioned SoxAX, the 40 kDa triheme cytochrome PufC in the photosynthetic reaction center (Alric et al., 2004), and the 15 kDa DsrJ in sulfate respiration (Pires et al., 2006) have the axial coordination. NaxL and NaxS have no homology to these proteins in the primary structure and the physiological roles of these proteins seem to be distinctly different. Nevertheless, the His/Cys coordination in heme c might commonly contribute to the protein functions.

One possibility of such a contribution is to create the very low redox potential of heme. The two hemes in a SoxA subunit of P. pantotrophus have low redox potentials: one is −432 mV and the other is lower than that (Reijerse et al., 2007). The heme c in DsrJ is also reported to have a low redox potential. The relatively high σ-donor ability of thiolate ligand, Cys-, effectively stabilizes the ferric state of heme, and conceivable polar surroundings would make the heme–iron redox potential further lower. Taken together, NaxLS of the anammox bacterium strain KSU-1 appears to be a novel member of c-type heme proteins with His/Cys axial coordination and a low redox potential.

The low redox potential of NaxLS reminds us of its potential role as an electron transmitter in anammox bacteria, in which electrons with a very low redox potential are generated on oxidation of hydrazine catalyzed by HZO and/or HAO, or on ferredoxin oxidation–reduction that is supposed to occur in anammox processes in C. Kuenenia stuttgartiensis (Strous et al., 2006). The physiological role of the NaxLS protein has not been elucidated as yet and further investigation is required.