Electrostatic attraction between cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> and cytochrome <Emphasis Type="Italic">c</Emphasis> affects kinetics of cytochrome <Emphasis Type="Italic">c</Emphasis> reduction

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.     

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.     

Interaction between uranium and the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>3</Subscript> of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> strain G20

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.     

Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

Cloning,expression, and physicochemical characterization of a new diheme cytochrome <Emphasis Type="Italic">c</Emphasis> from <Emphasis Type="Italic">Shewanella baltica</Emphasis> OS155

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV–vis, magnetic circular dichroism, and ¹H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid–base equilibria with pK _a values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of −0.144 and −0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye–Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°′.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Optimisation of cellobiose dehydrogenase production by the fungus<Emphasis Type="Italic"> Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>)<Emphasis Type="Italic"> rolfsii</Emphasis>

The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l(-1) (15,000 units cytochrome c activity l(-1)) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.     

Laue crystal structure of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase from a high-yield expression system

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “small tetraheme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).     

Molecular modeling and dynamics simulation of a histidine-tagged cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript>

Although an affinity tag such as six consecutive histidines, (His)₆-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)₆-tag was introduced to the N-terminus of a small heme protein, cytochrome b ₅, experimental results showed the resultant protein, (His)₆-cyt b ₅, has similar property and function to that of isolated cyt b ₅. To provide structural information for this observation, we herein performed a structural prediction of (His)₆-cyt b ₅ by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)₆-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b ₅ through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b ₅, which indicates that the N-terminal (His)₆-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)₆-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.     

Characterization of two cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript> proteins from the cyanobacterium <Emphasis Type="BoldItalic">Gloeobacter violaceus</Emphasis> PCC 7421

In the genome of the untypical cyanobacterium Gloeobacter violaceus PCC 7421 two potential cytochrome b ₆ proteins PetB1 and PetB2 are encoded. Such a situation has not been observed in cyanobacteria, algae and higher plants before, and both proteins are not characterized at all yet. Here, we show that both apo-proteins bind heme with high affinity and the spectroscopic characteristics of both holo-proteins are distinctive for cytochrome b ₆ proteins. However, while in PetB2 one histidine residue, which corresponds to H100 and serves as an axial ligand for heme b _H in PetB1, is mutated, both PetB proteins bind two heme molecules with different midpoint potentials. To recreate the canonical heme b _H binding cavity in PetB2 we introduced a histidine residue at the position corresponding to H100 in PetB1 and subsequently characterized the generated protein variant. The presented data indicate that two bona fide cytochrome b ₆ proteins are encoded in Gloeobacter violaceus. Furthermore, the two petB genes of Gloeobacter violaceus are each organized in an operon together with a petD gene. Potential causes and consequences of the petB and petD gene heterogeneity are discussed.     

Study of redox potential in cytochrome <Emphasis Type="Italic">c</Emphasis> covalently bound to terminal oxidase of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> FTU

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.     

Peculiarities of cyanide binding to the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript>-type cytochrome oxidase from the thermophilic bacterium <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Cytochrome c oxidase of the ba ₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a ₃ in cytochrome ba ₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a ₃²⁺-CN complex of cytochrome ba ₃ is strikingly tight. Experiments have shown that the K _d value of the cytochrome ba ₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10⁻⁸ M, which is four to five orders of magnitude less than the K _d of the cyanide complex of the reduced heme a ₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba ₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10⁻⁷ sec⁻¹), while the rate of binding (k _on ∼ 10² M⁻¹·sec⁻¹) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba ₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a ₃ does not depend on pH in the neutral area, but it approaches linear dependence on H⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK _a = 8.75.     

pH and CO<Subscript>2</Subscript> effects on <Emphasis Type="Italic">Coelastrella</Emphasis> (<Emphasis Type="Italic">Scotiellopsis</Emphasis>) <Emphasis Type="Italic">rubescens</Emphasis> growth and metabolism

We studied effects of рН and СО₂ enrichment on the physiological condition and biochemical composition of a carotenogenic microalga Coelastrella (Scotiellopsis Vinatzer) rubescens Kaufnerová et Eliás (Scenedesmaceae, Sphaeropleales, Chlorophyceae), a promising source of natural astaxanthin. The microalga was grown at a constant pH (5, 6, 7 or 8) maintained by direct СО₂ injection. The air-sparged culture served as the control. Cell division rate and size, dry biomass productivity, the rates of nitrogen and phosphorus uptake as well as photosynthetic pigment and total lipid content and fatty acid composition were followed. С. rubescens possessed a narrow-range рН tolerance (the optimum рН 6–7). Under these conditions, the highest values of the maximum (1.0–1.1 1/day) and average (0.3–0.35 1/day) specific growth rate, chlorophyll а (4.8–4.9%) and total carotenoid dry weight percentages (1.7–1.8%) were recorded. Cell lipid fatty acid unsaturation index (1.851) and polyunsaturated fatty acid percentage (36–39%) and С18:3 ω3/С18:1 ω9 ratio (3.8–4.5) were also the highest under these conditions. A decline of рН to 5 brought about severe stress manifesting itself as a cell division cessation, photosynthetic apparatus reduction, two-fold increase in cell volume, accumulation of dry weight and lipids and a considerable decline in fatty acid unsaturation. Cultivation of С. rubescens without СО₂ enrichment resulted in a rapid alkalization of the medium to рН 9.5–10.5 impairing the physiological condition of the cells. Reasons of the deteriorative effects of suboptimal pH values on the physiological condition of C. rubescens are discussed.     

Genetic diversity of the cytochrome b gene fragment haplotypes in red-backed vole <Emphasis Type="Italic">Myodes</Emphasis> (<Emphasis Type="Italic">Clethrionomys</Emphasis>) <Emphasis Type="Italic">rutilus</Emphasis> Pallas, 1779

For the first time, genetic analysis of the cytochrome b gene fragment haplotypes encoding the identical and the most common cytochrome b polypeptide (F1) in M. rutilus from eastern and Beringian maternal lineages was carried out. The F1 frequencies for the vole populations from Northern Priokhotye and the Kolyma basin were calculated. Considerable polymorphism of the cytochrome b F1 haplotypes within two major phylogroups of red-backed vole was supported by high molecular diversity indices for these clades. The proportion of genetic variation between the maternal lineages of F1 red-backed vole individuals (60.71%) was considerably higher than inter(24.44%) and intrapopulation (14.85%) components. The data obtained make it possible to advance a hypothesis on the convergence of the cytochrome b polypeptide structure upon sequence divergence of the corresponding gene.     

Cloning,Expression, and Characterization of Siamese Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>) Hemoglobin from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.     

Mediated catalysis of <Emphasis Type="Italic">Paracoccus pantotrophus</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase by <Emphasis Type="Italic">P. pantotrophus</Emphasis> pseudoazurin: kinetics of intermolecular electron transfer

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E ⁰′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 10⁵ M⁻¹ s⁻¹ was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.     

NMR-validated structural model for oxidized<Emphasis Type="Italic"> Rhodopseudomonas palustris</Emphasis> cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>556</Subscript>

The structure of oxidized Rhodopseudomonas palustris cytochrome c ₅₅₆ has been modeled after that of high-spin cytochrome c from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0511-2     

X-ray structure of methanol dehydrogenase from<Emphasis Type="Italic"> Paracoccus denitrificans</Emphasis> and molecular modeling of its interactions with cytochrome<Emphasis Type="Italic"> c</Emphasis>-551i

The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA). The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions. The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence. The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i). Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling. As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used. In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter. Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding. One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i. Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i. The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.