The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.     

Thermodynamically controlled supramolecular polymerization of cytochrome b562

A hemoprotein‐based supramolecular polymer that has a covalently linked heme moiety on the protein surface has been constructed based on interprotein heme–heme pocket interactions of the chemically modified apocytochrome b₅₆₂ ( 1 ‐H63C). The thermodynamic properties of the polymer have been investigated by means of size exclusion chromatography, UV–vis spectroscopy, and circular dichroism spectroscopy. The results indicate that, as with other synthetic systems reported so far, the 1 ‐H63C hemoprotein assembly is thermodynamically controlled in aqueous solution: the degree of polymerization is dependent on the 1 ‐H63C concentration and is modulated by the addition of the end‐capping units, native heme, and/or apocytochrome b₅₆₂ mutant (apoH63C). These properties suggest a potential use for the hemoprotein self‐assembly in preparation of stimuli‐responsive functional nanobiomaterials. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 194–200, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Crystallographic study of cytochrome b 562 from Escherichia coli

Three crystalline forms of cytochrome b₅₆₂ from Escherichia coli have been grown. One form, suitable for a three-dimensional structure investigation, is triclinic, containing two molecules per cell, with the following parameters: a = 33·71 Å, b = 50·51 Å, c = 32·71 Å, α = 102.62°, β = 86.67° and γ = 107.03°. X-ray data from the native crystals have been collected to 2.8 Å resolution and a search for suitable heavy atom derivatives has begun.     

The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.     

On the evolutionary relationship of the 4-alpha-helical heme proteins. The comparison of cytochrome b562 and cytochrome c'

The atomic models of the cytochrome b562 and cytochrome c' monomers have been compared. When the respective heme groups are superimposed, the four alpha-helices of each nearly coincide. Four aromatic side chains, including the heme ligands, and a methionine occur in spatially equivalent positions in contact with the heme groups. This structural evidence suggests that the two cytochrome families may have diverged from a common molecular ancestor.     

Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli.

The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b562 using the lac-promoter produced the protein to a level of 3-5% of total protein. This over-production enables employment of a simple, high-yield purification protocol to obtain homogeneous cytochrome b562. Spectroscopic and N-terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E. coli B [Itagaki, E. & Hager, L.P. (1966) J. Biol. Chem. 241, 3687-3695]. It is demonstrated that the genomic sequence codes for a classic N-terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space.     

Thermodynamic and spectroscopic characteristics of the cytochrome bc1 complex. Role of quinone in the behavior of cytochrome b562

Cytochrome b562 does not behave as a single independent thermodynamic component in preparations of purified quinol cytochrome c reductase. This effect is much more pronounced in quinone sufficient preparations; in such preparations, the epr spectrum of the cytochrome is Eh sensitive, with a peak shift from g = 3.42 to 3.48 occurring as the potential is lowered from 100 mV to 0 mV. The peak shift is dependent on the presence of quinone and can be restored to quinone-depleted preparations by supplementation with ubiquinol 2 if phospholipid depletion is not too severe. The results suggest that cytochrome b562 is strongly interacting with the Qc quinone binding site.     

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.     

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.     

The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b₅₆₂ in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92±0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b₅₆₂ is a component of the yeast SDH.     

Characterisation of a Rrhodobacter sphaeroides gene that encodes a product resembling Eescherichia coli cytochrome b(561) and R. sphaeroides cytochrome b(562)

Analysis of the photoactive yellow protein (pyp) gene region of Rhodobacter sphaeroides has revealed the presence of an additional open reading frame, orfD, that had not previously been identified. Here we report the location of this new gene and the predicted amino acid sequence of the encoded protein. The translation product resembles a group of small cytochrome b-like proteins, including Escherichia coli cytochrome b(561), R. sphaeroides cytochrome b(562), and two new cytochrome b(561)-like proteins identified using the E. coli genome sequence, for which functions have not yet been established. To determine OrfD function in R. sphaeroides, an orfD mutant was constructed. The OrfD mutant exhibited growth rates and yields very similar to those of the wild-type strain when grown under a variety of growth conditions. Respiration rates, reduced-minus-oxidised spectra and levels of photosynthetic complexes were also very similar in the two strains. Although the role of OrfD was therefore not determined here, we demonstrate that the orfD gene is expressed in R. sphaeroides under aerobic, semi-aerobic and photosynthetic growth conditions.     

Glucose enzyme electrode using cytochrome b(562) as an electron mediator

We demonstrate the construction of glucose sensors employing pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQGDH) from Acinetobacter calcoaceticus and glucose oxidase (GOD) from Aspergillus nigar coupled with Escherichia coli soluble cytochrome b(562) (cyt b(562)) as electron acceptor. PQQGDH and GOD do not show direct electrochemical recycling of the prosthetic group at the electrode surface leading to a corresponding current signal. We constructed PQQGDH and GOD electrodes co-immobilized with 100-fold molar excess of cyt b(562) and investigated the electrochemical properties without synthetic electron mediators. PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes both responded well to glucose whereas no current increase was observed from the electrode immobilizing enzyme alone. The detection limits for the PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes were 0.1 and 0.8 mM, respectively, and their linearity extended to over 2 and 9 mM, respectively. These results demonstrate that a sensor system can be constructed without a synthetic electron mediator by using a natural electron acceptor. Furthermore, we have demonstrated the potential application of cyt b(562) in direct electron transfer type sensor systems with oxidoreductases whose quaternary structure do not contain any electron transfer subunit.     

Coupled oxidation of heme covalently attached to cytochrome b562 yields a novel biliprotein

A variant of Escherichia coli cytochrome b(562) with covalently attached heme can be converted to a biliverdin-containing protein in two distinct stages by coupled oxidation and acid hydrolysis. The first stage of coupled oxidation yields a stable verdoheme-containing protein. This verdoheme protein is unusual in three respects. First, the verdoheme group is covalently bound to the protein through a c-type thioether linkage. Second, the oxidation stops at the verdoheme stage, and finally, this is the first report of verdoheme generated from a heme protein with exclusive methionine ligation to the heme iron. In addition, the oxidation process does not require denaturation of the protein. The product has been characterized by optical spectroscopy, ESI mass spectrometry, and (1)H NMR. The NMR data show that the predominant product is the result of oxidation at the alpha-meso carbon. A collective evaluation of data on the topic suggests that the electronic structure of the heme, not protein steric effects, is the main factor in controlling the regiospecificity of the oxidation site. In the second stage of conversion to a biliprotein, we demonstrate that the verdoheme ring can be opened by treatment with aqueous formic acid to give alpha-biliverdin covalently attached to the folded protein. This product, a protein-bound linear tetrapyrrole as characterized by optical spectroscopy and mass spectrometry, is an example of a phycobilin chromophore that has not been observed previously.     

Effect of redox potential of the heme on the peroxidase activity of cytochrome b562

Measurements of peroxidase activities of two site-specific mutants and wild type cytochrome b562 suggest that the enzymatic activity correlates with the redox potential of the metal center. A lower value of the Fe(3+)/Fe(2+) redox potential seems to be important for promoting peroxidase activity of the hemeprotein possibly by stabilization of the high-valent redox intermediate involved in the catalytic function. The results provide an approach towards rational tuning of enzyme function when 'grafted' into a new protein environment.     

A cytochrome b562 variant with a c-type cytochrome CXXCH heme-binding motif as a probe of the Escherichia coli cytochrome c maturation system

Cytochrome b562 is a periplasmic Escherichia coli protein; previous work has shown that heme can be attached covalently in vivo as a consequence of introduction of one or two cysteines into the heme-binding pocket. A heterogeneous mixture of products was obtained, and it was not established whether the covalent bond formation was catalyzed or spontaneous. Here, we show that coexpression from plasmids of a variant of cytochrome b562 containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins results in an essentially homogeneous product that is a correctly matured c-type cytochrome. Formation of the holocytochrome was accompanied by substantial production of its apo form, in which, for the protein as isolated, there is a disulfide bond between the two cysteines in the CXXCH motif. Following addition of heme to reduced CXXCH apoprotein, spontaneous covalent addition of heme to polypeptide occurred in vitro. Strikingly, the spectral properties were very similar to those of the material obtained from cells in which presumed uncatalyzed addition of heme (i.e. in the absence of Ccm) had been observed. The major product from uncatalyzed heme attachment was an incorrectly matured cytochrome with the heme rotated by 180 degrees relative to its normal orientation. The contrast between Ccm-dependent and Ccm-independent covalent attachment of heme indicates that the Ccm apparatus presents heme to the protein only in the orientation that results in formation of the correct product and also that heme does not become covalently attached to the apocytochrome b562 CXXCH variant without being handled by the Ccm system in the periplasm. The CXXCH variant of cytochrome b562 was also expressed in E. coli strains deficient in the periplasmic reductant DsbD or oxidant DsbA. In the DsbA- strain under aerobic conditions, c-type cytochromes were made abundantly and correctly when the Ccm proteins were expressed. This contrasts with previous reports indicating that DsbA is essential for cytochrome c biogenesis in E. coli.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.