The use of a water-soluble carbodiimide to study the interaction between Chromatium vinosum flavocytochrome c-552 and cytochrome c

The interaction between horse heart cytochrome c and Chromatium vinosum flavocytochrome c-552 was studied using the water-soluble reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Treatment of flavocytochrome c-552 with EDC was found to inhibit the sulfide: cytochrome c reductase activity of the enzyme. SDS gel electrophoresis studies revealed that EDC treatment led to modification of carboxyl groups in both the M_r 21000 heme peptide and the M_r 46000 flavin peptide, and also to the formation of a cross-linked heme peptide dimer with an M_r value of 42000. Both the inhibition of sulfide: cytochrome c reductase activity and the formation of the heme peptide dimer were decreased when the EDC modification was carried out in the presence of cytochrome c. In addition, two new cross-linked species with M_r values of 34000 and 59000 were formed. These were identified as cross-linked cytochrome c-heme peptide and cytochrome c-flavin peptide species, respectively. Neither of these species were formed in the presence of a cytochrome c derivative in which all of the lysine amino groups had been dimethylated, demonstrating that EDC had cross-linked lysine amino groups on native cytochrome c to carboxyl groups on the heme and flavin peptides. A complex between cytochrome c and flavocytochrome c-552 was required for cross-linking to occur, since ionic strengths above 100 mM inhibited cross-linking.     

Complex formation between Chlorobium limicola f. thiosulfatophilumc-type cytochromes

It has been possible to demonstrate, using affinity chromatography, that Chlorobium flavocytochrome c-553 forms an electrostatically stabilized complex with Chlorobium cytochrome c-555. The binding site for cytochrome c-555 appears to be located on the heme-containing subunit of flavocytochrome c-553. This complex appears to be involved in the flavocytochrome c-553-catalyzed transfer of electrons from sulfide to cytochrome c-555. Complex formation has also been demonstrated between Chlorobium cytochromes c-555 and c-551, two components involved in the oxidation of thiosulfate by this green sulfur bacterium. Affinity chromatography data also suggest the possibility that the cytochrome binding sites on the Chlorobium flavocytochrome c-553 and on flavocytochrome c-552 from the purple sulfur bacterium Chromatium vinosum may be similar.     

Location of a cytochrome c binding site on the surface of flavocytochrome b 2

Saccharomyces cerevisiae flavocytochrome b ₂ couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate constant for cytochrome c reduction by flavocytochrome b ₂ depends on the rate of complex formation and is sensitive to ionic strength. Mutations in the heme domain of flavocytochrome b ₂ (Glu63→Lys, Asp72→Lys and the double mutation Glu63→Lys:Asp72→Lys) have significant effects on the reaction with cytochrome c, implicating these residues in complex formation. This kinetic information has been used to guide molecular modelling studies, which are consistent with there being no one single best-configuration. Rather, there is a set of possible complexes in which the docking-face of cytochrome c can approach flavocytochrome b ₂ in a variety of orientations. Four cytochromes c can be accommodated on the flavocytochrome b ₂ tetramer, with each cytochrome c forming interactions with only one flavocytochrome b ₂ subunit. All the models involve residues 72 and 63 on flavocytochrome b ₂ but in addition predict that Glu237 may also be important for complex formation. These acidic residues interact with the basic residues 13, 27 and 79 on cytochrome c. Through this triangle of interactions runs a possible σ-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavocytochrome b ₂) and ends with the ring of Pro68, which is in van der Waals contact with the cytochrome c heme. In total, the edge-to-edge "through space" distance from the imidazole ring of His66 to the C3C pyrrole ring of cytochrome c is 13.1?Å.     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.     

Structural Evidence for the Functional Importance of the Heme Domain Mobility in Flavocytochrome b2

Yeast flavocytochrome b₂ (Fcb2) is an l-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b₅-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b₂, hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 Å resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2·cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b₅·cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b₅. The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.     

A hypothetical complex between crystalline flavocytochrome b2 and Cytochrome c

Flavocytochrome b₂ and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b₂ led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b₂. The b₂ and c hemes are coplanar, with an edge-to-edge distance of 14 Å. the contact surface area is ca. 800 Ų. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b₂ stabilize the binding of cytochrome c. © 1993 Wiley-Liss, Inc.     

Flavin-containing heme enzymes

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the l-lactate dehydrogenase flavocytochrome b₂ and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Electrochemical and infrared spectroscopic analysis of the interaction of the CuA domain and cytochrome c552 from Thermus thermophilus

The hydrophobically guided complex formation between the Cu_A fragment from Thermus thermophilus ba₃ terminal oxidase and its electron transfer substrate, cytochrome c₅₅₂, was investigated electrochemically. In the presence of the purified Cu_A fragment, a clear downshift of the c₅₅₂ redox potential from 171 to 111 mV ± 10 mV vs SHE′ was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

SERR Spectroelectrochemical Study of Cytochrome cd 1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c 552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd ₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd ₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd ₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd ₁) depends on the presence of its physiological redox partner, cytochrome c ₅₅₂ (cyt c ₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd ₁ and cyt c ₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd ₁ and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd ₁ // cyt c ₅₅₂ and Ag // SAM // cyt c ₅₅₂ // Mhcd ₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c ₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd ₁ undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd ₁, reinforcing the idea that subtle and very specific interactions between Mhcd ₁ and cyt c ₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd ₁.     

A study of the interaction between two proteins,one containing a flavin mononucleotide

Interaction between cytochrome c and flavocytochrome b₂ has been studied in presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Affinity of the probe to flavocytochrome b₂ increase when the complex between the two proteins is obtained. Binding of TNS increases the fluorescence of flavocytochrome b₂ FMN. When the stoichiometry of complex between the two proteins is reached, TNS looses its affinity and stops binding on the flavocytichrome b₂; consequently, FMN fluorescence increase is no more observed. The dissociation constant of the complex was found equal to 0.1 μM. A similar result was obtained for the interaction between cytochrome c and flavodehydrogenase domain. The latter was obtained by proteolysis of flavocytochrome b₂.     

The reaction of Euglena gracilis cytochrome c-552 with nonphysio-logical oxidants and reductants.

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c₂. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c₂.     

Electrochemically driven catalysis of Rhizobium sp. NT-26 arsenite oxidase with its native electron acceptor cytochrome c552

We describe the catalytic voltammograms of the periplasmic arsenite oxidase (Aio) from the chemolithoautotrophic bacterium Rhizobium sp. str. NT-26 that oxidizes arsenite to arsenate. Electrochemistry of the enzyme was accomplished using its native electron transfer partner, cytochrome c₅₅₂ (cyt c₅₅₂), as a mediator. The protein cyt c₅₅₂ adsorbed on a mercaptoundecanoic acid (MUA) modified Au electrode exhibited a stable, reversible one-electron voltammetric response at + 275 mV vs NHE (pH 6). In the presence of arsenite and Aio the voltammetry of cyt c₅₅₂ is transformed from a transient response to an amplified sigmoidal (steady state) wave consistent with an electro-catalytic system. Digital simulation was performed using a single set of parameters for all catalytic voltammetries obtained at different sweep rates and various substrate concentrations. The obtained kinetic constants from digital simulation provide new insight into the kinetics of the NT-26 Aio catalytic mechanism.     

Isolation and properties of membrane-bound cytochrome c-552 from photosynthetic bacterium Chromatium vinosum

A membrane-bound cytochrome c-552 was isolated and purified from the photosynthetic bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate and bacterial alkaline protease followed by gel filtration. The purified cytochrome c-552, which may have been modified by the protease treatment, was electrophoretically homogeneous. Its minimal molecular weight was estimated to be 19 and 20 kdaltons, respectively by SDS polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-100. Cytochrome c-552 showed the absorption maxima at 419, 523 and 552 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 10.6 for the wavelength pair, 552 minus 540 nm. The midpoint potential at pH 8.0 was ?130 mV. The polarity in the amino acid composition of cytochrome c-552 was 40.1% and reflected its hydrophobicity. The solubilized cytochrome c-552 was shown to be a different entity from the soluble flavocytochrome c-552 in several respects.     

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Characterization and expression of the co-transcribed cyc1 and cyc2 genes encoding the cytochrome c4 (c552) and a high-molecular-mass cytochrome c from Thiobacillus ferrooxidans ATCC 33020

The sequence of the cyc1 gene encoding the Thiobacillus ferrooxidans ATCC 33020 c₅₅₂ cytochrome, shows that this cytochrome is a 21-kDa periplasmic c₄-type cytochrome containing two similar monohaem domains. The kinetics of reduction and the fact that cytochromes c₄ are considered to be physiological electron donors of cytochrome oxidases suggest that the last steps of the iron respiratory chain are: rusticyanin→cytochrome c₄→cytochrome oxidase. In Thiobacillus ferrooxidans, cyc1 is co-transcribed with the cyc2 gene, encoding a high-molecular-mass monohaem cytochrome c. This suggests that the cytochromes encoded by these genes belong to the same electron transfer chain.     

Complex formation and electron transfer between mitochondrial cytochrome c and flavocytochrome c552 from Chromatium vinosum

Flavocytochrome c552 from Chromatium vinosum catalyzes the oxidation of sulfide to sulfur using a soluble c-type cytochrome as an electron acceptor. Mitochondrial cytochrome c forms a stable complex with flavocytochrome c552 and may function as an alternative electron acceptor in vitro. The recognition site for flavocytochrome c552 on equine cytochrome c has been deduced by differential chemical modification of cytochrome c in the presence and absence of flavocytochrome c552 and by kinetic analysis of the sulfide:cytochrome c oxidoreductase activity of m-trifluoromethylphenylcarbamoyl-lysine derivatives of cytochrome c. As with mitochondrial redox partners, interaction occurs around the exposed heme edge at the "front face" of cytochrome c. However, the domain recognized by flavocytochrome c552 seems to extend to the right of the heme edge, whereas the site of interaction with mitochondrial cytochrome c oxidase and reductase is more to the left. Km but not Vmax of the electron transfer reaction with mitochondrial cytochrome c increases with increasing ionic strength. The correlation of chemical modification and ionic strength dependence data indicates that the electrostatic interaction between the two hemoproteins involves fewer ionic bonds than that with other redox partners of cytochrome c.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.