The acidic domain of cytochrome c₁ in Paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc₁ complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c₅₅₂

Abstract

The cytochrome bc₁ complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c₁ subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c₅₅₂.

1. Introduction

Complex III (ubiquinol:cytochrome c oxidoreductase, or cytochrome bc₁ complex) catalyzes electron transfer from ubiquinol to cytochrome c coupled to proton translocation across the membrane according to the protonmotive Q-cycle originally proposed by Peter Mitchell [1]. The enzyme is a functional dimer, with each monomer composed of 9-11 polypeptide chains in mitochondria [2, 3] and 3-4 in bacteria [4, 5]. In the first step of the Q-cycle quinol is oxidized at the Q_o site in an electron bifurcation reaction [6]. The first electron is transferred from quinol to the Rieske iron-sulfur protein of the high potential chain, and the second electron is passed to heme b_L of the low potential chain. From the iron-sulfur center, electron transfer proceeds to cytochrome c₁ by domain movement of the entire globular head of the Rieske protein, and thereafter to cytochrome c. The second electron is transferred from heme b_L to heme b_H, which reduces quinone in the Q_i site to semiquinone. After the first hemicycle, one quinol has been oxidized at the Q_o site, extruding two protons to the intermembrane space. In the second hemicycle, another quinol is oxidized by the same bifurcation reaction, finally reducing the Q_i-site semiquinone to quinol concomitant with proton uptake from the cytosolic or matrix side. Overall, the cytochrome bc₁ complex reaction can be expressed as:

$${\mathrm{QH}}_2+2\mathrm{cyt}.c^{3+}+2{{\mathrm{H}}^{+}}_{\mathrm{N}}\to \mathrm{Q}+2\mathrm{cyt}.c^{2+}+4{{\mathrm{H}}^{+}}_{\mathrm{p}}$$

where the indices P and N denote the positive and negative sides of the membrane. In this study we investigate the cytochrome bc₁ complex from Paracoccus denitrificans, encoded by the fbc operon (Fig. 1) [7]. The Rieske iron sulfur protein (ISP), product of fbcF, carries the [2Fe-2S] cluster. FbcB encodes cytochrome b, containing the two b-type hemes. Cytochrome c₁, the product of fbcC, houses a covalently attached c-type heme, and sequence analysis showed the presence of three different regions in FbcC: an N-terminal, strongly acidic domain, the heme binding core domain, and the C-terminal hydrophobic region encoding a transmembrane helix anchoring the protein to the membrane. Early investigations [7, 8] revealed the unique composition of the acidic domain with 40% acidic residues, 40% alanines and 18% prolines. In this stretch no basic residue is found, thus showing a high analogy to the acidic domain of Qcr6p from yeast or the hinge protein from bovine heart, suggesting a similar function. For the eukaryotic counterparts a role in cytochrome c interaction was proposed [9-16]. In addition these acidic domains of yeast and P. denitrificans complex III (on the Qcr6p and on the cytochrome c₁, resp.) are expected at exactly the same position in the X-ray structures of the respective complexes¹.

Figure 1. Schematic representation of the wild type subunits of the cytochrome bc₁ complex (left) and the cytochrome c₁ mutant complex (right) on SDS-PAGE isolated from P. denitrificans.

In red, the domain structure of the cytochrome c₁. On the right side, the deletion variant results in a lower band with an apparent molecular weight of about 30 kDa; the other subunits are not affected. (Two bands are visible for the ISP, most likely due to different conformers because of incomplete denaturation, a feature often observed for the P. denitrificans bc₁ complex.)

Cytochrome c₅₅₂ is the electron shuttle between complex III and the cytochrome aa₃ oxidase in P. denitrificans [17, 18], as confirmed by experiments using antibodies and deletion strains. Under specific solubilisation conditions [19, 20], supercomplexes with high electron transfer rates were isolated, containing complexes I, III and IV in a 1:4:4 ratio. Based on its DNA sequence, three domains have been identified for the cytochrome c₅₅₂ [7, 21]: an N-terminal, hydrophobic anchor, a flexible, negatively charged linker region, and a C-terminal heme liganding domain. The latter has been cloned [22], heterologously expressed in E. coli and its structure determined [23, 24]; this fragment (cytochrome c_552F) is used in this investigation as an electron acceptor.

To identify the role of the acidic domain in the P. denitrificans cytochrome c₁ subunit and to study the interaction between cytochromes c₁ and c₅₅₂ in the three subunit complex, the entire acidic domain of cytochrome c₁ of 150 amino acids has been deleted [25], and the mutant complex expressed and purified. The complex of the truncation mutant is a dimer and is structurally fully intact, as shown by its X-ray structure at 2.7 Å resolution¹.

Here we analyze this truncation variant by steady-state kinetic and ruthenium flash photolysis experiments, showing wild type properties in its kinetic parameters and no changes in apparent substrate interaction, which rules out any involvement of the cytochrome c₁ acidic domain in the electron transfer process.

1. Materials and Methods

1.1 Materials

Dodecylmaltoside (DDM) was obtained from Merck (Darmstadt). Horse heart cytochrome c and decylubiquinone were purchased from Sigma, and the latter reduced as described before and quantified by UV-spectroscopy using reported extinction coefficients [26, 27].

1.2 Cloning procedures

The construction of the His tagged deletion mutant of the Paracoccus denitrificans bc₁ complex has been obtained by in-frame restriction of the cytochrome c₁ gene (XhoI, NotI), followed by nuclease treatment and ligation. Deletion of the deca His-tag was achieved by the use of Splicing by Overlap Extension PCR (SOEing PCR), as already described [28].

1.3 Expression

The expression vector containing the truncated version of the fbc operon was transferred to a P. denitrificans strain (MK6, [29]) and the cells selected on plates containing rifampicin, kanamycin and streptomycin.

Paracoccus denitrificans cytochrome c_552F was expressed and purified as described previously [22].

1.4 Purification of Cytochrome bc₁ Complex

Cells from an overnight growth were harvested at an OD value of 3-5 and resuspended in a buffer containing 100 mM sodium phosphate, pH 8, and 1 mM EDTA, and frozen. Membranes were obtained as described in [28]. To solubilise the bc₁ complex membranes were diluted to a total protein concentration of ca. 35 mg/ml. The solution was 1:1 diluted with the solubilisation buffer (100 mM MES/NaOH pH 6, 600 mM sucrose, 2.4 M NaCl for the wild-type; 50 mM sodium phosphate buffer pH 8, 600 mM sucrose, 2.4 M NaCl for the His-tagged wild-type and the deletion mutant) and DDM was added to a weight ratio of 1.2:1 (detergent:protein). The solution was stirred on ice for one hour, then centrifuged for one hour (>100,000 x g), and the supernatant was diluted to a final concentration of 350 mM NaCl before loading it on the ion exchange column in the wild-type and the wild type His tagged case. The deletion mutant complex was purified using affinity chromatography (Ni²⁺-NTA, Qiagen), in a buffer containing 50 mM NaP_i pH 8, 300 mM NaCl, 0.02% DDM, and eluted with buffers containing imidazole or histidine (gradients from 10-250 mM or 0-200 mM respectively). Fractions were collected and analysed by redox difference spectra, SDS PAGE and Western Blot, in order to assess the purity of the sample and its protein composition. Protein concentrations were calculated using the extinction coefficient of 56 mM⁻¹ cm⁻¹ at 560-574 nm for the two hemes in cytochrome b [30]. Cytochrome c_552F purification was done as described in [22].

1.5 Steady-State Reduction of bc₁ Complexes

Steady-state kinetics were recorded in an Hitachi spectrophotometer in 50 mM MOPS/NaOH pH 7.5, 100 mM NaCl, 0.04% DDM, 1 mM EDTA, 1 mM KCN, using decylubiquinol as substrate. The reduction of cytochrome c from horse heart was followed at 550 nm. For P. denitrificans cytochrome c_552F, the reduction was followed at 552 nm. In a disposable cuvette, varying amounts of cytochrome c or c_552F (from 1 μM to 25 μM) were mixed with 80 μM decylubiquinol and an appropriate amount of purified bc₁ complex. For each cytochrome c concentration, measurements were performed in triplicate and initial rates calculated, using an extinction coefficient of 21.5 mM⁻¹ cm⁻¹ for the horse heart cytochrome c [28] and 19.4 mM⁻¹ cm⁻¹ for the cytochrome c_552F [17]. The kinetic parameters k_CAT and K_M were calculated according to the Michaelis-Menten equation using Origin 8.0.

1.6 Flash photolysis experiments

Rapid kinetic experiments were carried out using two ruthenium-labeled cytochrome c derivatives, Ru_z-N23C-c_552F and Ru_z-H39C-Cc, prepared as described in [21]. Ru_z-N23C-c_552F is the P. denitrificans cytochrome c_552F with the surface Asn²³ mutated to Cys (N23C) and covalently attached to Ru_z. Ru_z-H39C-Cc is yeast isocytochrome c with Cys 102 mutated to threonine (C102T) and a surface cysteine introduced in position 39 (H39C), covalently attached to Ru_z [21]. Ru_z is Ru(2,2′-bipyrazine)₂(4-bromomethyl-4′-methyl-2,2′-bipyridine). The flash photolysis experiments were carried out using a phase R model DL 1400 flash lamp-pumped dye laser and a detection system described by Heacock et al. [31]. Solutions contained about 5 μM Ru_z-N23C-c_552F or Ru_Z-H39C-Cc and 5 μM cytochrome bc₁ in 300 μl of Tris/HCl buffer (20 mM in the case of Ru_Z-H39C-Cc and 10 mM for Ru_z-N23C-c_552F) pH 8.0 with 0.02% dodecylmaltoside in semimicro glass cuvettes at 10 ° C. 2 mM sodium ascorbate, and 2 μM N, N, N’, N’-tetramethylphenylendiamine (TMPD) were added to fully reduce the heme groups, as verified by recording the visible spectra before flash photolysis. In the photooxidation experiments with Ru_z-H39C-Cc, [Co(NH₃)₅Cl]²⁺ was added (5 mM) as the sacrificial acceptor (Fig. 2). In photooxidation experiments with Ru_z −N23C-c_552F, pentaammineosmium nitrile ([Os(NH₃)₅(CH₃CN)](CF₃SO₃)³⁺, 1 mM) was used as the sacrificial acceptor. The ET reaction was monitored at 550 nm for Ru_Z-H39C-Cc and 552 nm for Ru_z-N23C-c_552F, and 557 nm (isosbestic for the two cytochromes, in order to observe the cytochrome c₁ oxidation kinetics). All absorbance transients were analyzed using the KINFIT kinetics program obtained from Online Instrument System Inc. Absorbance spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer.

Figure 2. Model of the high potential electron transfer chain of the bc₁ complex with cytochrome c as occurring in the laser flash photolysis measurements.

In the experiment, the cytochrome bc₁ complex and the Ru-labeled cytochrome c are pre-reduced due to the presence of ascorbate and TMPD. After flashing, the photo-excited Ru transfers one electron to the sacrificial oxidant in solution (red arrow 1), creating an electron vacancy, filled by the electron present on the electron acceptor cytochrome (red arrow 2). The electron present on the cytochrome c₁ is now transferred to the Ru-labeled cytochrome c (red arrow 3). Since the iron sulphur cluster is also reduced by ascorbate in solution, the cytochrome c₁ is then reduced again by the electron from the ISP (red arrow 4). The blue arrow symbolizes the movement of the ISP head domain. (The low potential chain does not participate in the electron transfer, since ascorbate cannot reduce the b-type hemes in cytochrome b.)

3. Results

3.1 Characterization of the deletion mutant of the Paracoccus denitrificans bc₁ complex

A mutant cytochrome bc₁ complex with a deletion of the N-terminal acidic domain in the cytochrome c₁ subunit was originally described by Gerhus [25]. A His-tag has been added via SOEing PCR [32, 33] at the C-terminal end of the cytochrome b subunit. This allowed efficient purification of the deletion mutant by metal affinity chromatography. Classical ion exchange chromatography used for the wild-type [34] did not give satisfactory results for the mutant, where the predominantly interacting acidic domain was deleted. The presence of the three subunits (ISP, cytochrome b and cytochrome c₁) was confirmed by SDS PAGE (Fig. 1). The initial purification protocol was carried out using imidazole to elute the protein, however with unexpected results. The reducibility of the cytochrome c₁ heme by ascorbate was strongly diminished, as well as the turnover number (results not shown). A similar effect has been described for complex III of Rhodobacter capsulatus [35], where high imidazole concentrations diminished the activity drastically (20 fold lower than wild-type) and impaired the reducibility of cytochrome c₁ by displacing of the heme liganding Met [36, 37]. Changing the elution buffer from imidazole to histidine kept the redox properties of cytochrome c₁ intact and gave turnover numbers comparable to the wild-type enzyme (see below).

3.2 Steady-state kinetics in presence of horse heart cytochrome c and P. denitrificans cytochrome c_552F

To investigate whether the acidic domain has any effect on cytochrome c reduction (cytochrome c_552F or cytochrome c_hh) by complex III, steady-state kinetics were measured. In the case of horse heart cytochrome c, the k_CAT and the Michaelis constant do not show significant changes between the wild-type and the mutant complex, indicating that the latter shows wild type kinetic parameters. Similar results were obtained with the physiological substrate, cytochrome c_552F (Table 1).

Table 1. Kinetic parameters from Michaelis-Menten kinetics measured under steady-state conditions of the three cytochrome bc₁ complexes with horse heart cytochrome c and cytochrome c_552F.

Kinetics have been measured in 50 mM MOPS/NaOH pH 7.5, 100 mM NaCl, 0.04% DDM, 1 mM EDTA, 1 mM KCN, using decylubiquinol as substrate. The reduction of cytochrome c from horse heart was followed spectroscopically at 550 nm. For P. denitrificans cytochrome c_552F, the reduction at 552 nm was followed. k_CAT and K_M were calculated using the Michaelis-Menten equation and fitted in Origin 8.0. Each substrate concentration has been measured three times to get statistical relevant values.

	Horse heart cytochrome c		Cytochrome c552F
	kCAT (s−1)	KM (μM)	kCAT (s−1)	KM (μM)
Wild-type	273 ± 25	2.3 ± 0.5	251 ± 13	2.7 ± 0.3
His-tagged wild-type	328 ± 22	3.4 ± 0.3	322 ± 25	4.8 ± 0.9
His-tagged deletion mutant	357 ± 19	3.1 ± 0.3	315 ± 13	3.8 ± 0.2

3.3 Pre steady-state analysis of the interaction between the bc₁ complexes from P. denitrificans and two different substrates

Fast kinetic measurements were performed in order to define the mode of interaction between cytochrome c₁ and cytochrome c₅₅₂. The reaction of the isolated bc₁ complex with two different substrates, Ru_z-N23C-c_552F and Ru_z-H39C-Cc were analyzed [21]. The ruthenium flash photolysis method was used to study the rapid electron transfer reactions between each of the cytochrome bc₁ preparations and Ru_z-H39C-Cc in the forward, physiological direction (Fig. 2). Laser flash photolysis of a solution containing 5 μM reduced wild-type complex III and 5 μM reduced Ru_z-H39C-Cc in 20 mM Tris/HCl, pH 8.0 led to a rapid decrease in the absorbance at 550 nm, indicating fast oxidation of Ru_z -H39C-Cc by photoexcited Ru_Z (II*) (Fig. 3). This was followed by an increase in the 550 nm absorbance transient due to electron transfer from cytochrome c₁ to cytochrome c. As a control, oxidation of cytochrome c₁ was detected at 557nm, isosbestic for Ru_z-H39C-Cc. The absorbance transient recorded at 550 nm for the wild-type complex was biphasic with a fast phase of 16,600 ± 2000 s⁻¹ and a small slow phase. The transient at 557 nm was a single exponential trace with a rate constant of 16,700 ± 2000 s⁻¹ (see Table 2). The slow phase in the 550 nm transient is due to the slow re-reduction of photooxidized Ru_z-H39C-Cc by ascorbate and TMPD, for it is not present in the traces recorded at 557 nm. The fast phase corresponds to the intramolecular electron transfer between cytochrome c₁ and cytochrome c when the two reaction partners are associated in a stable complex at a low ionic strength.

Figure 3. Transient traces and Brønsted plot for the reactions between the three cytochrome bc₁ complexes of P. denitrificans and its two different electron acceptors.

Panel A (top): Transient traces under the following conditions: 5 μM of each of the cytochrome bc₁ complexes with 5 μM Ru_z-H39C-Cc in 20 mM Tris/HCl pH 8.0, with 1 mM ascorbate, 2 μM TMPD, 5 mM [Co(NH₃)₅Cl]²⁺ and 0.02 % dodecylmaltoside. The black line is the transient recorded at 550 nm (Ru_z-H39C-Cc reduction), and the grey one at 557 nm (cytochrome c₁ oxidation). Panel B (top): Transient trace of each cytochrome bc₁ complex with 9 μM Ru_z-N23C-c_552F in 10 mM Tris/HCl pH 8.0, with 1 mM ascorbate, 2 μM TMPD, 1 mM penta-amineosmium and 0.02 % dodecyl-maltoside. In all cases, solutions were made anaerobic by purging with N₂ and maintained at 10 °C. In the lower panel A: Brønsted plot showing the dependency of the electron transfer rate on the salt concentration, indicating intramolecular electron transfer between the cytochrome bc₁ complex and the Ru_z-H39C-Cc below 90 mM I in solution. At higher I, the rate starts to diminish, giving similar Brønsted parameter for all three complexes, indicating no involvement of the acidic domain in the reaction. Lower panel B: Brønsted plot with the Ru_z-N23C-c_552F substrate. This indicates that with I values lower than 40 mM, a complex of much lower stability than in the previous case is formed. Also here, no difference among the samples in the interaction with Ru_z-N23C-c_552F is observed.

Table 2. Flash photolysis initiated electron transfer rates between the bc₁ complex isolated from P. denitrificans and Ru_z-H39C-Cc and Ru_z-N23C-c_552F.

The rates at 550 nm indicate the reduction of Ru_z-H39C-Cc or Ru_z-N23C-c_552F, whereas at 557 nm the oxidation of cytochrome c₁. The error is ± 20 %. For Ru_z-N23C-c_552F no 557 nm value was calculated as the recorded signal was too low for representative analysis, due to the fast re-reduction of cytochrome c₁ by the ISP (results not shown).

Ruz-H39C-Cc	WT	WT His-tagged	His-tagged deletion mutant
550 nm	16,600 s−1	9,300 s−1	14,600 s−1
557 nm	16,700 s−1	8,400 s−1	11,200 s−1
Ruz-N23C-c 552F
550 nm	1,900 s−1	1,500 s−1	1,000 s−1

As the salt concentration is increased, the electron transfer rate remains the same until an ionic strength of 90 mM is reached (Fig. 3). With rising I, the rate constant decreases, indicating dissociation of the complex and bimolecular electron transfer between Ru_Z-H39C-Cc and cytochrome bc₁. Electron transfer kinetics for wild-type, wild-type His-tagged, and deletion mutant complexes are largely comparable when assayed in 20 mM Tris/HCl buffer and 0.02 % dodecylmaltoside, as summarized in Table 2. The effect of ionic strength on the reactions of Ru_z-H39C-Cc with each of the three cytochrome bc₁ preparations is very similar, as shown in Figure 3. The Brønsted plots [38] of log k vs. the square root of ionic strength are nearly linear above 90 mM ionic strength. The Z_AZ_B Brønsted parameters (Table 3) indicate that the electrostatic interaction of Ru_z-H39C-Cc with each of the three cytochromes bc₁ preparations is similar.

Table 3. Z_AZ_B Brønsted parameters for the three complexes used in this study.

The values are comparable among the complexes in each set of experiments, indicating no major change in the total number of charged residues involved in the electron transfer reaction.

	Ruz-H39C-Cc	Ruz-N23C-c552F
wild-type	−5.57 ± 0.5	−2.78 ± 0.08
His-tagged wild-type	−5.68 ± 0.4	−3.36 ± 0.26
deletion mutant	−4.59 ± 0.3	−3.35 ± 0.17

The ruthenium flash photolysis method was also used to study the rapid electron transfer reactions between each of the cytochrome bc₁ preparations and the endogenous P. denitrificans substrate, Ru_z-N23C-c_552F in the forward, physiological direction. The 552 nm transient for the reaction of 5 μM wild-type cytochrome bc₁ complex with 6 μM Ru_z-N23C-c_552F in 10 mM Tris/HCl, pH 8.0 was 1,900 s⁻¹, indicating electron transfer from cytochrome c₁ to Ru_z-N23C-c_552F. The Brønsted plots did not give clear evidence for the formation of a stable complex between the reaction partners at low ionic strength. The rate constants for the other two cytochrome bc₁ complexes were similar, as shown in Table 2, and also decreased with increasing ionic strength, as shown in Figure 3. The Z_AZ_B Brønsted parameter are listed in Table 3 and show that the electrostatic interaction of Ru_z-N23C-c_552F with each of the three cytochromes bc₁ species is similar but distinctly lower when compared to the yeast cytochrome substrate.

4. Discussion

The cytochrome bc₁ complex from P. denitrificans is comprised of the three essential subunits carrying redox cofactors. However, as a unique feature compared to other organisms, its cytochrome c₁ subunit carries an extra, highly acidic domain at its N-terminus, preceding the canonical c-heme binding domain followed by its C-terminal membrane anchor. This acidic domain, although not homologous in sequence, mimics the acidic domain of the Qcr6p subunit in yeast, and the hinge protein in the mammalian complex. Comparison of the P. denitrificans truncation complex with the yeast complex III structure reveals both acidic domains positioned at the same area close to the cytochrome c₁ core domain¹. It has been suggested [11, 13] that this Qcr6p subunit may be involved in preorienting the cytochrome c substrate. The presence of the acidic domain in the cytochrome c₁ subunit of P. denitrificans, a commonly used model organism for the mitochondrial electron transfer chain, raised the question whether the same might hold true for this bacterial domain.

Janzon et al. [21] addressed electron transfer reactions of soluble fragments derived from cytochrome c₁ and cytochrome c₅₅₂ as well as point mutants in the c₁ heme binding cleft, in order to define the interactions between the two partners. In that work, it was suggested that the acidic domain does not contribute to the cytochrome c₁-c₅₅₂ interaction, and therefore it is important to study the situation in a different experimental scenario, with fully assembled complexes. In our work, the intact complex is studied, addressing not only the interactions between the two partner proteins, but also the consequences of the deletion in the cytochrome c₁ subunit. Moreover, this experimental setup more closely reflects the genuine situation in the membrane (see also below), but using the purified components. Structural observations in yeast [16, 39, 40] proposed that initial interactions occur via long-range orientation of these partner proteins, followed by hydrophobic contacts on the protein surfaces. Intracomplex electron transfer rates independent of ionic strength were reported for the yeast and the bovine complexes below 120 mM and 80 mM ionic strength, respectively [41]. Similar observations have been made for the soluble fragments in the P. denitrificans case [21].

The rate constant for the reaction of Ru_z-H39C-Cc with wild-type bc₁ complex is 16,600 s⁻¹ at low ionic strength, and remains independent of ionic strength up to 90 mM (Figure 3). This is consistent with electron transfer within a complex between Ru_z-H39C-Cc and bc₁, as observed previously for the soluble cytochrome c₁ domain [21]. At ionic strength values above 90 mM the rate constant decreases, indicating dissociation of the complex and bimolecular reaction between the dissociated partners. Interestingly, both the intracomplex and the bimolecular rate constants for the reaction of Ru_z-H39C-Cc with the entire wild-type cytochrome bc₁ is 3-fold smaller than that of the reaction with the soluble fragment of cytochrome c₁ [21]. The fully assembled membrane complex is much bulkier than the soluble fragments, since not just a domain but the whole obligate dimer or the tetramer are present in the truncation variant and the wild type complex, respectively¹ [42]. This might lead to a higher number of non-productive collisions between complex III and its substrate, explaining to some extent the lower rate constants. Under true physiological conditions, contraints for a successful encounter between the two domains must be assessed somewhat differently. While the bc₁ complex surface is largely shielded by being membrane embedded, the electron acceptor cytochrome c₅₅₂ is linked to the membrane suface by its N-terminal anchor sequence, thus effectively restricting its diffusional space. With the observation of supercomplexes in the electron transfer chain of P. denitrificans [19], a further diffusional restriction is most likely encountered.

The rate for the reaction of Ru_z-N23C-c_552F with wild-type bc₁ is much slower than that of Ru_z-H39C-Cc, and decreases with increasing ionic strength much earlier (Figure 3). This is an indication of a much less stable complex formed between Ru_z-N23C-c_552F and the soluble cytochrome c₁. Ionic strength-dependent kinetics of the reactions of both Ru_z-H39C-Cc and Ru_z-N23C-c_552F with the His-tagged deletion variant are comparable to the kinetics with wild-type cytochrome bc₁ (Figure 3). Both the respective rate constants and the Brønsted Z_AZ_B parameters are similar to the kinetic parameters for the wild-type enzyme. This indicates that the acidic domain in the P. denitrificans bc₁ complex does not play any significant role in the formation of the physiological electron transfer complex nor the actual electron transfer reaction. This is in contrast to other systems reported in the literature where the acidic subunit of the eukaryotic complex actually seems to be involved in electron transfer between the two proteins [15, 16].

The electron transfer chain of P. denitrificans is not only operative under aerobic conditions, but uses also nitrogen oxides (nitrate, nitrite, NO and N₂O) as terminal electron acceptors, making the cytochrome bc₁ complex an important electron hub involving other cytochromes c at this branch point in electron transfer [18, 43]. It may be speculated that the acidic domain plays a role in the interaction with other c-type cytochromes. The cytochrome bc₁ complex could be involved in distributing electrons to alternative metabolic pathways and providing fast adaptation to changing environmental conditions.

In the steady-state kinetic analysis, no substantial differences were observed between the three complexes using either the horse heart cytochrome c or the endogenous cytochrome c_552F. Neither k_CAT nor K_M were affected by the deletion of the acidic domain. This confirms the results obtained with the flash photolysis experiments, and together with the structural characterization of Kleinschroth et al.¹ clearly demonstrates that the deletion mutant of the P. denitrificans bc₁ complex is a valid model system also for kinetic studies as presented earlier [28]. The crystal structure of the complex reveals that the fold of the cytochrome c₁ subunit is not affected by the lack of this large domain¹ (Fig. 4). The oligomeric state, on the other hand, changes drastically, since the wild type complex is arranged as a “dimer of dimers” [42], whilst the variant is the obligate dimer¹. This kinetic characterization of the complex, revealing full wild type properties of the deletion variant together with the now available structural information, makes the P. denitrificans deletion complex an even more valuable model system for the mitochondrial counterparts.

Figure 4. X-ray structure at 2.7 Å resolution of the dimeric P. denitrificans cytochrome bc₁^Δac complex, revealing an intact cytochrome c binding site.

The complex is depicted as C_α trace with secondary structure elements. Cofactors and the inhibitor stigmatellin (STG) are shown in black stick-and-ball representation. Identical subunits in the dimer are coloured for cytochrome b in red/salmon, cytochrome c₁ in blue/marine and ISP in green/lime. The superimposed soluble domain of subunit cytochrome c₁ from yeast (3CX5; in cyan) allows a direct comparison of the undistorted cytochrome c binding site (red ellipse). The complex is viewed parallel to the membrane with the cytoplasmic side at the bottom.

Highlights.

The role of a unique acidic domain in the c₁ subunit of complex III is addressed

In a deletion variant, neither k_CAT nor K_M are changed

Laser flash photolysis indicates same substrate interaction in the variant complex

Abbrevations

ISP

iron sulphur protein

cytochrome c_hh

cytochrome c from horse heart

DDM

dodecylmaltoside

cytochrome bc₁^Δac complex

truncation variant of the P. denitrificans complex III