Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Production and preliminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurreducens in Escherichia coli

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c₇ from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c₇ and expressed it in Escherichiacoli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c₇ determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c₇ of known structure.     

Cytochrome c1 exhibits two binding sites for cytochrome c in plants

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c₁, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c₁ and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.     

Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography

A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc₁, was achieved using a short, 50 mm C₁₈-reversed-phase column. The short column decreased the elution time 4–7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Redox-coupled proton transfer in the active site of cytochrome cbb3

Cytochrome cbb₃ is a distinct member of the superfamily of respiratory heme-copper oxidases, and is responsible for driving the respiratory chain in many pathogenic bacteria. Like the canonical heme-copper oxidases, cytochrome cbb₃ reduces oxygen to water and couples the released energy to pump protons across the bacterial membrane. Homology modeling and recent electron paramagnetic resonance (EPR) studies on wild type and a mutant cbb₃ enzyme [V. Rauhamäki et al. J. Biol. Chem. 284 (2009) 11301-11308] have led us to perform high-level quantum chemical calculations on the active site. These calculations bring molecular insight into the unique hydrogen bonding between the proximal histidine ligand of heme b₃ and a conserved glutamate, and indicate that the catalytic mechanism involves redox-coupled proton transfer between these residues. The calculated spin densities give insight in the difference in EPR spectra for the wild type and a recently studied E383Q-mutant cbb₃-enzyme. Furthermore, we show that the redox-coupled proton movement in the proximal cavity of cbb₃-enzymes contributes to the low redox potential of heme b₃, and suggest its potential implications for the high apparent oxygen affinity of these enzymes.     

Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c₃ isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc1 function in vivo

Electronic connection between Q_o and Q_i quinone catalytic sites of dimeric cytochrome bc₁ is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc₁-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.     

Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).     

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc₁ complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc₁ complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 °C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Å resolution.     

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc₁ complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.     

Isolation and preliminary characterization of new cytochrome c from autotrophic haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

New small cytochrome c (TniCYT) was purified from haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. The protein was analyzed by mass spectrometry as well as using visible, CD and EPR spectroscopy. It was found that TniCYT is a monomer with a molecular mass of 9461 Da which contains two hemes per molecule. The data of CD and EPR spectroscopy showed that two hemes possess different optical activity and are in distinct, high and low spin states. TniCYT was also demonstrated to have unusual characteristics in the visible spectrum, namely, the splitting of characteristic peaks was observed in α- and β-bands of the heme spectrum when the reduced form of cytochrome was analyzed. The α-band has two peaks with maximum at 548 and 556 nm whereas the β-band showes ones at 520 and 528 nm. According to the MALDI finger-print analysis, the new cytochrome has a unique amino acid sequence.     

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E_550-535) of 19.7 ± 6.3 mM⁻¹ cm⁻¹ and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 ± 122 μmol min⁻¹ mg⁻¹ protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.     

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation

The effect of Zn²⁺ on the rates of electron transfer and of voltage generation in the cytochrome bc₁ complex (bc₁) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn²⁺ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b_h (in the absence of Zn²⁺ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c₁ reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn²⁺ <100 μM the reduction of heme b_h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc₁ turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b_h is the main electrogenic reaction in the cytochrome bc₁ complex, (2) imply an allosteric link between the site of heme b_h oxidation and the site of cytochrome c₁ reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc₁ complex.     

Redox-controlled backbone dynamics of human cytochrome c revealed by N NMR relaxation measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D ¹⁵N NMR relaxation experiments. ¹⁵N T₁ and T₂ values and ¹H-¹⁵N NOEs of uniformly ¹⁵N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S²), the effective correlation time for internal motion (τ_e), the ¹⁵N exchange broadening contributions (R_ex) for each residue, and the overall correlation time (τ_m) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S² value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S² values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.