cbb₃-type cytochrome oxidase in the obligately chemolithoautotrophic Thiobacillus sp. W5

Abstract

A highly active cytochrome c oxidase has been purified 75-fold from the neutrophilic obligately autotrophic Thiobacillus sp. W5. UV/visible and electron paramagnetic resonance spectroscopy revealed that the cytochrome c oxidase contains low-spin hemes c and low- and high-spin hemes b. HPLC analysis confirmed the presence of heme b as the sole type of non-covalently bound heme. The combined data from atomic absorption spectroscopy and electron paramagnetic resonance indicate the absence of Cu_A and suggest the presence of a bimetallic heme-copper redox center. These results show that Thiobacillus sp. W5 contains a cbb₃-type oxidase, which is a member of the heme–copper oxidase family. The cbb₃-type oxidase was the only cytochrome oxidase expressed in aerobically and micro-aerobically grown Thiobacillus sp. W5 cultures.

Heme–copper oxidase, Thiobacillus, Obligate chemolithoautotrophy, cbb₃-type oxidase

1 Introduction

Obligately chemolithoautotrophic thiobacilli play an important role in the sulfur cycle and are responsible for the biological oxidation of reduced sulfur compounds [1]. The ability of these bacteria to oxidize reduced sulfur compounds at very high rates has led to their use in biotechnological applications. These include waste water-treatment systems in which neutrophilic obligately autotrophic Thiobacillus species remove reduced sulfur compounds by partially oxidizing them to elemental sulfur [2]. The cytochromes and respiratory chains of the neutrophilic, obligately autotrophic thiobacilli have been the subject of study in several laboratories [3, 4] but, until now, the nature and structure of the terminal oxidases have been obscure.

Most oxidases of bacterial respiratory systems are members of the heme–copper oxidase superfamily, which include cytochrome c and quinol oxidases [5]. All of these enzymes possess a highly hydrophobic subunit with about 12 membrane-spanning α-helices, homologous to subunit I of the mammalian cytochrome c oxidase [6]. This subunit contains a low-spin heme, a high-spin heme and one copper (Cu_B), of which the latter two form a bimetallic centre at which site reduction of oxygen coupled to proton translocation occurs [7]. Recent work has divided the cytochrome oxidases into two groups [8]. The first group contains the cytochrome c oxidases, of which the second subunit has a mixed-valence binuclear Cu_A redox center [9]. This copper-containing subunit is involved in the binding of cytochrome c and serves as the initial electron acceptor. The second group of cytochrome c oxidases is formed by the cbb₃-type oxidases, which have only recently been recognised as members of the heme–copper superfamily. These oxidases lack a homologue of subunit II and do not contain Cu_A. Instead, a subunit containing cytochrome c is believed to function as the initial electron acceptor [10].

This study describes the partial purification and identification of the terminal cytochrome c oxidase from the obligately chemolithoautotrophic Thiobacillus sp. W5. This new Thiobacillus species was isolated as the dominant organism from a sulfur-producing waste water-treatment system. Taxonomically it was closely related to T. neapolitanus (Visser et al. unpublished results).

2 Materials and methods

2.1 Organism and cultivation

Thiobacillus sp. W5 (LMD 94.73) was obtained from the Delft Culture Collection. The organism was cultured in a chemostat under sulfide-limited conditions as described previously [11]. Two different oxygen regimes (Dissolved Oxygen Tensions (DOT) of 50% and 2% air saturation) were imposed. These are referred to as ‘aerobic’ and ‘micro-aerobic’, respectively.

2.2 Cytochrome c oxidase activity

Cytochrome c oxidase activity was assayed by measuring oxygen uptake in the presence of an electron donor using a Clark-type electrode at 25°C. The 3 ml reaction mixture contained 3.3 mM ascorbate, 333 μM TMPD and a preparation of cytochrome c oxidase in 50 mM Tris-HCl (pH 7.5).

2.3 Purification of the cytochrome c oxidase

Cells (75 g wet weight), collected at 4°C from a sulfide-limited chemostat (DOT 50%), were suspended in 200 ml 50 mM Tris-HCl (pH 7.5) and passed 3 times through a French pressure cell at 110 MPa. The broken cells were treated with DNase, after which whole cells and cell debris were removed by centrifuging at 10.000×g for 30 min. The membrane fraction was obtained by centrifuging at 200.000×g for 4 h. The membranes were then washed and resuspended in 50 mM Tris-HCl buffer (pH 7.5). The membrane proteins were solubilized for 1 h at 4°C by the addition of 1%β-d-lauryl maltoside. After centrifuging at 200.000×g for 6 h, the supernatant was loaded on a DEAE-Sepharose column (5×40 cm), previously equilibrated with 0.025% lauryl maltoside, 50 mM Tris-HCl, pH 7.5 (buffer A). Proteins were eluted with a linear gradient (1000 ml) from 0 to 0.5 M KCl in buffer A. Active fractions were concentrated (Centricon 30, Amicon), desalted (PD10, Pharmacia) and applied to a Mono Q column (10/10, Pharmacia), previously equilibrated with buffer A. The oxidase was eluted with a linear gradient (60 ml) from 0 to 0.5 M KCl in buffer A. Active fractions were concentrated and further purified by gel filtration on a Superdex 200 column (Pharmacia), previously equilibrated with 0.2 M KCl in buffer A.

2.4 UV/visible spectroscopy

UV/visible absorption spectra were made with a SLM Aminco DW-2000 spectrophotometer.

2.5 Heme analysis

The heme c and b content of the purified sample were determined simultaneously from available pyridine hemochrome spectra [12]. Identification of the non-covalently linked hemes was carried out by HPLC, using a Nova Pak C₁₈ reversed-phase column (3.9×150 mm) [13]. Bovine heart cytochrome c oxidase (aa₃; Sigma) and horse myoglobin (heme b; Serva) were used as references.

2.6 Protein analysis

Protein was determined according to Bradford [14] and the BCA method (Pierce Chemical Co.)

2.7 SDS-PAGE analysis

SDS-PAGE electrophoresis was done at room temperature according to Laemmli [15] using Mini Protean equipment (BioRad). Gels were stained for protein with Coomassie Brilliant Blue G250. Heme staining was done with 3,3′,5,5′-tetramethylbenzidine and H₂O₂[16].

2.8 Electron paramagnetic resonance

Electron paramagnetic resonance (EPR) spectroscopy was done using a Varian E-9 spectrometer operating at X-band frequency (9.2 GHz) at 11 K. The sample contained approximately 50 μM air-oxidized cytochrome c oxidase, which was flushed with argon to remove oxygen prior to freezing.

2.9 Metal analysis

Copper was determined by furnace Atomic Absorption Spectroscopy (AAS) using the Standard Addition Technique at the Laboratory of Material Sciences of Delft University of Technology.

3 Results

3.1 Purification and subunit composition of the cytochrome c oxidase

The results of each step of the purification of the cytochrome c oxidase are shown in Table 1. A 75-fold purification was finally obtained after a 5-step procedure. SDS-PAGE analysis revealed the presence of four major protein bands at 45, 40, 38 and 28 kDa. Some impurities, with molecular weights above 70 kDa, were also observed. Heme staining revealed that the 45, 38 and the 28 kDa bands contained covalently bound heme c. Attempts to further purify the cytochrome oxidase resulted in total loss of activity, which could not be reconstituted.

1

Purification of cytochrome c oxidase from Thiobacillus sp. W5

Purification step	Protein	Activity	Recovery	Specific activity	Purification
(mg)	(μmol min−1)	(%)	(μmol mg−1 min−1)	(-fold)
Crude extracts	2068	6330	100	3.1	1.0
Membranes	973	5460	86	5.6	1.9
Detergent extraction	589	4940	78	8.4	2.8
DEAE-Sepharose	98	1920	30	19.6	6.5
Mono Q	9	940	15	104.4	34.8
Gel filtration	3	665	10	221.7	73.9

UV/visible absorption spectra of cell free extracts from Thiobacillus sp. W5 cells, grown at dissolved oxygen tensions of 50 and 2%, revealed no differences, indicating that the cytochrome composition of the respiratory chain was not affected by the external oxygen concentration. In line with this observation, a small-scale purification of the cytochrome c oxidase from micro-aerobically grown Thiobacillus sp. W5 cells yielded an oxidase preparation with identical properties.

3.2 Heme and metal composition

The dithionite-reduced minus ferricyanide-oxidized difference spectrum of the purified oxidase gave maxima at 422 nm (γ-band), 524 nm (β-band) and 552 nm (α-band), with a distinct shoulder at 560 nm, indicating low-spin heme c and b in the cytochrome c oxidase complex (Fig. 1). Ascorbate/PMS was able to fully reduce the cytochromes present in the complex, indicating that these cytochromes are all high-potential cytochromes. The lack of absorbance near 600 nm shows that heme a is not present. The air-oxidized minus air-oxidized plus cyanide difference spectrum showed a peak at 404 nm and a through at 419 nm (Fig. 2). The spectroscopic changes, caused by the binding of cyanide to the oxidized enzyme, suggest the presence of a high-spin heme b.

1

UV/visible absorption spectra of the purified cytochrome oxidase from Thiobacillus sp. W5. Dithionite-reduced spectrum (———); ferricyanide-oxidized spectrum (–––); reduced minus oxidized difference spectrum (- - -).

2

Air-oxidized minus air-oxidized plus CN difference spectrum of the purified cytochrome oxidase of Thiobacillus sp. W5.

Matrix analysis of the mixture of pyridine hemochromes showed a heme c content of 22.6±1.1 nmol (mg protein)⁻¹ and a heme b content of 9.5±1.0 nmol (mg protein)⁻¹, indicating a heme c/heme b ratio of 5:2. AAS revealed 4.9±0.7 nmol Cu (mg protein)⁻¹. Assuming a molecular mass of 106 kDa for the cytochrome c oxidase, a specific heme and copper content of 2.4±0.1 mol heme c (mol oxidase)⁻¹, 1.0±0.1 mol heme b (mol oxidase)⁻¹ and 0.5±0.1 mol copper (mol oxidase)⁻¹ could be calculated. The specific copper and heme b content do not meet the minimal need for 1 copper and 2 hemes b (high and low spins) per oxidase. These differences might be caused by impurities in the preparation. When the heme content is normalized to a minimum copper content of 1 mole per mole of oxidase, a copper/heme b/heme c ratio of 1:2:4.8 was obtained.

The nature of the non-covalently bound hemes was investigated by reverse-phase HPLC of the extracted hemes from purified cytochrome c oxidase (Fig. 3). Extracted hemes b from myoglobin and a from cytochrome oxidase aa₃ were used as references. Hemes o and a are more hydrophobic than heme b because of a farnesylhydroxyethyl side chain, resulting in longer retention times. The non-covalently bound hemes from the purified cytochrome c oxidase proved to be of the b-type.

3

Reverse-phase analysis of extracted hemes from the cytochrome oxidase of Thiobacillus sp. W5 (A) and horse myoglobin (heme b) and bovine heart cytochrome aa₃ oxidase (B). The retention times of heme b and heme a were 6.0 and 13.5 min, respectively.

3.3 Electron paramagnetic resonance

An EPR spectrum of the purified enzyme is shown in Fig. 4. The characteristic spectral features of Cu_A[9] are absent. A broad (g=3.04) and sharp (g=2.99) EPR signal, with corresponding g_x,y values around g=2.24 and g=1.49, indicate low-spin hemes. The signal with g_z= 3.404 is from a highly anisotropic low-spin heme b[17]. A small and substoichiometric amount of high-spin heme signal (g=6.0) is also observed. The signal with g=2 is from an unknown radical.

4

X-band EPR spectrum of oxidized cytochrome oxidase from Thiobacillus sp. W5. Numbers above the signals indicate g-values. Experimental conditions: frequency, 9.234 GHz; modulation amplitude, 1.6 mT; microwave power, 0.2 mW; temperature, 11 K.

3.4 Catalytic properties

The catalytic activity of the purified cytochrome c oxidase was measured as oxygen consumption with ascorbate/TMPD as electron donors. It was calculated that 560 mol oxygen was consumed per mole of oxidase per second. Addition of 25 μM cyanide completely inhibited the reaction.

4 Discussion

This paper reports the 75-fold purification and identification of a cytochrome c oxidase Thiobacillus sp. W5. The highly active cytochrome c oxidase preparation resisted complete purification as further chromatography led to almost complete and irreversible inactivation. This cytochrome c oxidase is probably a cbb₃-type oxidase because: (1) HPLC analysis showed that only heme b was present as the non-covalently bound heme in the cytochrome oxidase; (2) UV/visible absorption and EPR spectroscopy revealed the presence of low-spin hemes c and b and high-spin heme b, while a-type hemes could not be detected; (3) AAS revealed the presence of copper in the oxidase; (4) EPR spectroscopy showed that a Cu_A-specific signal was not present. Moreover, the absence of a stoichiometric amount of copper and high-spin heme may indicate that these groups form an anti-ferromagnetically coupled bimetallic redox center.

SDS-PAGE analysis showed the presence of four major protein bands, indicating four subunits. Three of these subunits had covalently bound heme. This is in contrast to the three subunits present in the cbb₃-type oxidases of Rhodobacter capsulatus[18], R. sphaeroides[10] and Paracoccus denitrificans[19], only two of which had covalently bound heme. Further study is necessary to identify the nature of the fourth protein band and confirm that it is indeed a subunit or an impurity.

cbb₃-type oxidases have recently been shown to be new members of the heme–copper oxidase superfamily. They have been found in R. sphaeroides[10], R. capsulatus[18], P. denitrificans[19] and Bradyrhizobium japonicum[20]. All of these organisms express the cbb₃ oxidase when grown under anaerobic or micro-aerobic conditions. At high dissolved oxygen concentrations, cytochrome aa₃, which has a lower affinity for oxygen, is expressed. Thiobacillus sp. W5, however, seems to express cytochrome cbb₃ as its sole cytochrome oxidase under both aerobic and micro-aerobic conditions. This might be due to the fact that the Thiobacilli often occur at the oxic/anoxic interface, where low concentrations of sulfide and oxygen can coexist. Preliminary experiments have shown that the phylogenetically related T. neapolitanus also may possess a cbb₃-type of oxidase and does not have a cytochrome aa₃-type oxidase.

Acknowledgements

This work (Project: IMB 91208) was financially supported by the IOP on Environmental Biotechnology which is financed by the Ministry of Economic Affairs and the Ministry of Housing, Physical Planning and the Environment.