Cytochromes b₅₆₁ are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four ...different transmembrane α-helices. Although cytochromes b₅₆₁ residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b₅₆₁ has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the Nδ₁ atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.
Cytochromes b₅₆₁, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our ...recent studies on recombinant Zea mays cytochrome b₅₆₁ suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b₅₆₁ as well and that conserved Lys⁸³ on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg⁷² and Tyr⁷¹. Removal of a positive charge at Arg⁷² did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr⁷¹ with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr⁷¹ and Arg⁷² with ascorbate. However, their mechanistic roles were distinctly different from that of Lys⁸³, as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.
Cytochromes b₅₆₁ constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane α-helices and two haem groups. Of particular ...interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b₅₆₁ heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b₅₆₁ with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His⁸⁶, His¹⁵⁹ and Lys⁸³, leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem centres did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b₅₆₁ conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H⁺/e⁻ transfer mechanism and that the specific N-carbethoxylation of haem axial His⁸⁶ that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys⁸³ might be important for an initial step(s) of the fast electron acceptance from ascorbate.
Cytochrome b561 family was characterized by the presence of “b561 core domain” that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two ...heme
b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants
+
DoH), and G (SDR2). In group A, both motif 1, {FN(X)HP(X)
2M(X)
2G(X)
5G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, {LFSWHP(X)
2M(X)
3F(X)
3M(X)EAIL(X)SP(X)
2SS}, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X)
3Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X)
2G(R/Y)} ,were found in group F at different regions from those of motifs 1 and 2. The “DoH” domain common to the NH
2-terminal region of dopamine β-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561.
Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our ...recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys83 on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg72 and Tyr71. Removal of a positive charge at Arg72 did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr71 with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr71 and Arg72 with ascorbate. However, their mechanistic roles were distinctly different from that of Lys83, as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.
Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our ...recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71). Removal of a positive charge at Arg(72) did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr(71) with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr(71) and Arg(72) with ascorbate. However, their mechanistic roles were distinctly different from that of Lys(83), as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.
Cytochromes b(561), a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic ...transmembrane alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys(83) might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.
