Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.     

1H-NMR investigation of yeast cytochrome c. Interaction with the corresponding specific reductase (L-lactate cytochrome)

1H-NMR spectroscopy has been used to study the modifications of certain characteristic resonances of the Hansenula anomala yeast cytochrome c on binding to its specific reductase (flavocytochrome b2) or to the isolated cytochrome domain obtained from the entire molecule. Normal titration curves are observed for the resonances at 37.8 ppm assigned to heme c methyl 8 and at 19.4 ppm, line of cytochrome b2 spectrum. In contrast, the shifts near 3.2 and 3.4 ppm for trimethyl-lysine resonances of this cytochrome c present abnormal titration curves, saturation being apparently reached at low molar (cytochrome b2)/(cytochrome c) ratio. An interpretation is proposed in terms of shifts due to local conformational transitions induced by reductase binding but not rapidly reversible upon dissociation.     

Crystallographic study of the recombinant flavin-binding domain of Baker's yeast flavocytochrome b(2): comparison with the intact wild-type enzyme

Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.     

Transient kinetics of the one-electron transfer reaction between reduced flavocytochrome b2 and oxidized cytochrome c. Evidence for the existence of a protein complex in the reaction

The one-electron transfer reaction from reduced flavocytochrome b2 (fully reduced by three electron equivalents) to ferricytochrome c, both purified from the yeast Hansenula anomala, has been studied using stopped-flow spectrophotometry in the course of a single turnover, for reactants initially mixed in a heme molar ratio equal to one. The cytochrome c reduction proceeded to completion through an apparently first-order process. Depending on the experimental conditions (concentrations and or ionic strength), the reduction is of second-order or first-order character. To interpret these kinetic results computer simulation studies have been performed based on a kinetic scheme involving, besides the formation of a complex before the electron transfer step, intramolecular electron transfer steps within flavocytochrome b2 to maintain the concentration of the specific electron donor center, the reduced cytochrome b2. As far as the cytochrome c reduction rate constant, ka, and its variations were concerned the simulated data showed that this complicated scheme could approximate a mechanism which is by far the simplest, involving only the two former steps. Such a scheme accounts firstly for the hyperbolic dependence of the rate of reduction of cytochrome c, ka, upon reductant concentrations which had provided clear evidence for the kinetic existence of a complex in the reaction pathway. At 5 degrees C the rate constant for the electron transfer is 380 s-1 with an activation energy of 13.8kJ mol-1 (3.3 kcal mol-1). Secondly it predicts the observed variations of ka with ionic strength and provides estimates of the rate constants of the binding step.     

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence.

The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = -1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.     

L-Lactate cytochrome c reductase: rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.     

Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 X 10(7) M-1 S-1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c', and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-alpha-S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b5.     

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.     

Inhibition of L-lactate: cytochrome-c reductase (flavocytochrome b2) by product binding to the semiquinone transient. Loss of reactivity towards monoelectronic acceptors

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39-45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J. M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491-503]. In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs-pyruvate complex is actually much more stable than the Fo-pyruvate and Fh-pyruvate complexes. The inhibition produced by pyruvate has been characterized under steady-state conditions using both ferricytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead-end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.     

Proteolytic cleavage of Hansenula anomala flavocytochrome b2 into its two functional domains. Isolation of a highly active flavodehydrogenase and a cytochrome b2 core

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of 'gene fusion'.     

The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "b5-like" heme. The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, J Mol Biol 212:837-863). The heme domains protrude from the central body of the tetramer consisting of the 4 FMN binding domains. Because only 2 heme domains are visible in the electron density map, the other 2 are probably disordered. We crystallized the Escherichia coli recombinant flavocytochrome b2 from S. cerevisiae inhibited by sulfite. Although the crystals were obtained under very different conditions from those of the pyruvate-containing native enzyme, they were found to be isostructural (P 3(2) 2 1, a = b = 164.5 A, c = 114.0 A). The 2.6-A X-ray structure was extensively refined with X-PLOR (R = 17.3%), which made it possible to describe in detail the recombinant flavocytochrome b2 molecular structure. There exist few differences between the native and recombinant structures, in line with the fact that they show similar kinetic behavior, and they further confirm the intrinsic mobility of the heme domain (Labeyrie F, Beloil JC, Thomas MA, 1988, Biochim Biophys Acta 953:134-141). This structure will be used as a starting model in the structural resolution of flavocytochrome b2 point mutants.     

Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2. Kinetic study of the inactivation reaction.

Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors. Inactivation was irreversible. The behaviour of both forms of flavocytochrome b2 (lintact and proteolytically cleaved) was examined. It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction. The value of the lactate dissociation constant of intact cytochrome b2 cytochrome b2 was determined in competition experiments with bromopyruvate. By comparison with the divergent published values for the Ks of the cleaved from, it appears that only those that differ from the Km by a factor of two or three are reasonable. This study opens the way for the identification of an active site residue and localization in the peptide chain of the bifunctional enzyme.     

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.     

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.     

Binding of Cibacron Blue F3GA to flavocytochrome b2 from baker's yeast.

1. Flavin-free cytochrome b2 has been prepared by rapid Sephadex filtration at acid pH. The method, which yields an apo-enzyme with high reconstitution potential and has several advantages over previously used procedures, is described in detail. 2. Flavin-free cytochrome b2 thus prepared is retained by blue-dextran-bound Sepharose. It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN. The holoenzyme is not retarded at all. 3. Both flavin-free and holocytochrome b2 bind Cibacron blue F3GA with appearance of distinct difference spectra. Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate. 4. It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b2. Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor. Taken together these results may mean that the enzyme possesses a local flavin-binding structure similar to the 'dinucleotide fold'.     

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.     

Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

Fluorescence studies on the interaction between two cytochromes extracted from the yeast, Hansenula anomala

The binding of cytochrome c to the cytochrome b2 core, both extracted from the yeast, Hansenula anomala, has been studied. Cytochrome b2 core heme is extracted and replaced by the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). A dissociation constant in the range of 85 microM is found for the TNS-apoprotein complex with a stoichiometry of 1:1. The interaction between the two proteins is followed by monitoring changes in the TNS fluorescence. We find the interaction between the cytochrome c and the apocytochrome b2 core to be dependent upon the ionic strength. The dissociation constant of this complex at 20 mM ionic strength is 6 +/- 2 microM with a 1:1 stoichiometry. This dissociation constant is similar to that estimated, by other researchers, for the dimer Zn cytochrome c-cytochrome b2 core complex.