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 c(3) 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.     

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.     

Distance dependence of interactions between charged centres in proteins with common structural features

Data collected for interactions among redox centres, and interactions between redox centres and acid-base residues in a family of small multihaem cytochromes are analysed. The distance dependent attenuation of the interactions between non-surface charges, for separations that range from 8 to 23 angstroms, can be described by a simple function derived from the Debye-Huckel formalism, fit to 9.5 and 7.6 as values for the relative dielectric constant and Debye length, respectively. However, there is considerable scatter in the data despite the structural similarities among the proteins, which is discussed in the framework of using such simple models in predicting properties of novel proteins.     

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

Type I cytochrome c(3) 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 c(3). This work presents the NMR assignment of the haem substituents in type I cytochrome c(3) isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c(3) 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.     

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.     

Homotropic and heterotropic interactions in cytochromes c3 from sulphate reducing bacteria

This article reviews the contribution of António Xavier and his associates to the study of multihaem cytochromes. Detailed analysis of interactions between the charges of the haems and protonatable groups reveals that electrostatic effects may be outweighed by subtle changes in conformation. This helps us to understand how tetrahaem cytochromes c₃ work and also gives an insight into the mechanism of cytochrome c oxidase.     

The tetraheme cytochrome from Shewanella oneidensis MR-1 shows thermodynamic bias for functional specificity of the hemes

Bacteria of the genus Shewanella contain an abundant small tetraheme cytochrome in their periplasm when growing anaerobically. Data collected for the protein isolated from S. oneidensis MR-1 and S. frigidimarina indicate differences in the order of oxidation of the hemes. A detailed thermodynamic characterization of the cytochrome from S. oneidensis MR-1 in the physiological pH range was performed, with data collected in the pH range 5.5–9.0 from NMR experiments using partially oxidized samples and from redox titrations followed by visible spectroscopy. These data allow the parsing of the redox and redox–protonation interactions that occur during the titration of hemes. The results show that electrostatic effects dominate the heme–heme interactions, in agreement with modest redox-linked structural modifications, and protonation has a considerable influence on the redox properties of the hemes in the physiological pH range. Theoretical calculations using the oxidized and reduced structures of this protein reveal that the bulk redox–Bohr effect arises from the aggregate fractional titration of several of the heme propionates. This detailed characterization of the thermodynamic properties of the cytochrome shows that only a few of the multiple microscopic redox states that the protein can access are significantly populated at physiological pH. On this basis a functional pathway for the redox activity of the small tetraheme cytochrome from S. oneidensis MR-1 is proposed, where reduction and protonation are thermodynamically coupled in the physiological range. The differences between the small tetraheme cytochromes from the two organisms are discussed in the context of their biological role.     

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.     

Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidans.

Trihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.     

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.     

Cytochrome c-551.5 (c7) from Desulfuromonas acetoxidans.

Cytochrome c-551.5 of the anaerobic sulfur-reducing bacterium Desulfuromonas acetoxidans has been purified to homogeneity and characterized. It elicits absorption bands at 551.5, 522.5 and 418 nm in the reduced form; the absorptivity ratio Aalpha(red)/A280nm(ox) equals 3.8 for the pure preparation. The molecular weight was estimated to be 9800 by gel filtration. Determination of the amion acid composition and analysis of the N-terminal amino acid sequence showed the cytochrome to be identical with the threehaem cytochrome c-551.5 (c7) isolated from the syntrophic mixed culture Chloropseudomonas ethylica strain 2K. The occurrence of multihaem cytochromes c in bacteria is discussed.     

Redox-Coupled Protonation of Respiratory Complex I: The Hydrophilic Domain

Respiratory complex I, NADH:ubiquinone oxidoreductase, is a large and complex integral membrane enzyme found in respiring bacteria and mitochondria. It is responsible in part for generating the proton gradient necessary for ATP production. Complex I serves as both a proton pump and an entry point for electrons into the respiratory chain. Although complex I is one of the most important of the respiratory complexes, it is also one of the least understood, with detailed structural information only recently available. In this study, full-finite-difference Poisson-Boltzmann calculations of the protonation state of respiratory complex I in various redox states are presented. Since complex I couples the oxidation and reduction of the NADH/ubiquinone redox couple to proton translocation, the interaction of the protonation and redox states of the enzyme are of the utmost significance. Various aspects of complex I function are presented, including the redox-Bohr effect, intercofactor interactions, and the effects of both the protein dielectric and inclusion of the membrane.     

Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability.

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.     

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy

We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten-halogen light source. Emergent light in the visible region (from 490-650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a 'steady-state' baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.     

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.     

Modeling electron transfer thermodynamics in protein complexes: interaction between two cytochromes c(3)

Redox protein complexes between type I and type II tetraheme cytochromes c(3) from Desulfovibrio vulgaris Hildenborough are here analyzed using theoretical methodologies. Various complexes were generated using rigid-body docking techniques, and the two lowest energy complexes (1 and 2) were relaxed using molecular dynamics simulations with explicit solvent and subjected to further characterization. Complex 1 corresponds to an interaction between hemes I from both cytochromes c(3). Complex 2 corresponds to an interaction between the heme IV from type I and the heme I from type II cytochrome c(3). Binding free energy calculations using molecular mechanics, Poisson-Boltzmann, and surface accessibility methods show that complex 2 is more stable than complex 1. Thermodynamic calculations on complex 2 show that complex formation induces changes in the reduction potential of both cytochromes c(3), but the changes are larger in the type I cytochrome c(3) (the largest one occurring on heme IV, of approximately 80 mV). These changes are sufficient to invert the global titration curves of both cytochromes, generating directionally in electron transfer from type I to type II cytochrome c(3), a phenomenon of obvious thermodynamic origin and consequences, but also with kinetic implications. The existence of processes like this occurring at complex formation may constitute a natural design of efficient redox chains.     

Spectral components of the α-band of cytochrome oxidase

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.     

Comparison of the redox reactions of various types of cytochrome c with iron hexacyanides

The dynamic behavior of various types of cytochromes c in the redox reaction with iron hexacyanides was studied using a temperature-jump method in order to elucidate the molecular mechanism of the redox reaction of cytochromes with their oxidoreductants. Transmittance after the temperature jump changed through a single exponential decay for all cytochromes investigated. Under a constant concentration of anion, the redox reaction of various types of cytochrome c with iron hexacyanides was analyzed according to the scheme: (see formula in text) where C(III) and C(II) are ferric and ferrous cytochromes, respectively, Fe(III) and Fe(II) are ferri- and ferrocyanides, respectively, C(III) . Fe(II) is the ferricytochrome-ferrocyanide complex and C(II) . Fe(III) is the ferrocytochrome-ferricyanide complex. When step B is slower than the other two steps A and C, tau-1 can be represented approximately as (see formula in text) where the bar over the variables denotes the equilibrium value. In a large excess of ferrocyanide against cytochrome, we can estimate kappa 2, kappa-2, K1 and K3 independently. In the case of horse cytochrome c at 18 degrees C in 0.1 M phosphate buffer at pH 7 with 0.3 M KNO3, the estimated parameters are kappa 2 = 100 +/- 50 S-1, kappa-2 = (3.5 +/- 1.0) . 10(3) S-1, K1 = 15 +/- 7 M-1 and K3 = (8.5 +/- 1.5). 10(-4) M. From the same experiments for seven cytochromes (cytochrome c from horse, tuna, Candida krusei, Saccharomyces oviformis, Rhodospirillum rubrum cytochrome c2, Spirulina platensis cytochrome c-554 and Thermus thermophilus cytochrome c-552), the following results can be deduced. (1) Each parameter defined in the scheme above (kappa 2, kappa-2, K1, K3) diverged beyond the error range. Above all, kappa 2 values of cytochromes c-554 and c-552 are as large as 1 . 10(4) S-1 and much larger than those for the other cytochromes (to 50 approx. 700 S-1). (2) The variance of kappa 2K1 and kappa-2/K3 are relatively less than the variances of individual parameters (kappa 2, kappa-2, K1 and K3), which suggests that the values of kappa 2K1 and kappa-2/K3 have been conserved during the course of evolution.     

Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c(3) isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.