Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports extravesicular dopamine beta-hydroxylase activity

Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine beta-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine beta-hydroxylase.     

Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer

Cytochromes b(561) 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 alpha-helices. Although cytochromes b(561) 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 b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) 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.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.     

Mapping the electron transfer interface between cytochrome b5 and cytochrome c

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.     

The structure of cytochrome b561, a secretory vesicle-specific electron transport protein

Cytochrome b561 is a transmembrane electron transport protein that is specific to a subset of secretory vesicles containing catecholamines and amidated peptides. This protein is thought to supply reducing equivalents to the intravesicular enzymes dopamine-beta-hydroxylase and alpha-peptide amidase. We have purified cytochrome b561 from bovine adrenal chromaffin granules by reverse phase chromatography and have determined internal amino acid sequences from peptides. Complementary oligonucleotides were used to isolate two cDNA clones from a bovine brain library. The structure predicted by the sequences of these cDNAs suggests a highly hydrophobic protein of 273 amino acids which spans the membrane six times with little extramembranous sequence. Cytochrome b561 is not homologous to any other cytochrome and thus represents a new class of electron carriers. RNA blotting experiments indicate that cytochrome b561 is expressed in the adrenal medulla and all brain regions of the cow, but not in visceral organs. This result agrees well with the putative function of this unique cytochrome and with the notion that this protein is localized to large dense-core synaptic vesicles.     

Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes

Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. The time course of the reduction of oxidized cytochrome b(561) with ascorbate could not be fitted with a single exponential but with a linear combination of at least four exponential functions. This result is consistent with the notion that cytochrome b(561) contains two hemes b, each having a distinct redox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. The fastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on the extravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequential ordered mechanism. The result showed K(s) = 2.2 mM for ascorbate at pH6.0. At a region below pH5.5, there was a significant lag before the reduction of hemes b occurred. This time lag was interpreted as due to a pH-dependent transient state before the first electron transfer to take place. The fastest phase was completely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) and Lys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibited the N-carbethoxylation of the histidyl residues and, thereby, restored the final reduction level of hemes b. But the reduction rate was still only one-twentieth of the native form. This result suggested an important role of the conserved Lys85 for the interaction with ascorbate.     

Photoinduced electron transfer reaction from N-formyl-L-kynurenine and L-kynurenine to cytochrome C

The reduction of cytochrome c was found in the presence of N-formyl-L-kynurenine (NFK) and L-kynurenine (KN) during irradiation, suggesting electron transfer to cytochrome c. The reaction occurred both under aerobic and anaerobic conditions. In the former case, oxygen molecules may act mainly as a quencher of excited NFK and KN, and superoxide anion produced by electron transfer may partially contribute to the reduction. The reaction proceeded via the excited triplet state of NFK and KN. The actual reductive chemical species might be an intermediate from excited state NFK or KN, which is assumed to be ketyl radical type species.     

Planarian cytochrome b(561): conservation of a six transmembrane structure and localization along the central and peripheral nervous system

Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.     

Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses

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 NH2-terminal region of dopamine beta-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.     

Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b561

Cytochrome b(561) mediates equilibration of the ascorbate/semidehydroascorbate redox couple across the membranes of secretory vesicles. The cytochrome is reduced by ascorbic acid and oxidized by semidehydroascorbate on either side of the membrane. Treatment with diethyl pyrocarbonate (DEPC) inhibits reduction of the cytochrome by ascorbate, but this activity can be restored by subsequent treatment with hydroxylamine, suggesting the involvement of an essential histidine residue. Moreover, DEPC inactivates cytochrome b(561) more rapidly at alkaline pH, consistent with modification of a histidine residue. DEPC does not affect the absorption spectrum of cytochrome b(561) nor does it change the midpoint reduction potential, confirming that histidine modification does not affect the heme. Ascorbate protects the cytochrome from inactivation by DEPC, indicating that the essential histidine is in the ascorbate-binding site. Further evidence for this is that DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a reaction mechanism in which ascorbate loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.     

Role of specific lysine residues in binding cytochrome c2 to the Rhodobacter sphaeroides reaction center in optimal orientation for rapid electron transfer

The role of specific lysine residues in facilitating electron transfer from Rhodobacter sphaeroides cytochrome c2 to the Rb. sphaeroides reaction center was studied by using six cytochrome c2 derivatives each labeled at a single lysine residue with a carboxydinitrophenyl group. The reaction of native cytochrome c2 at low ionic strength has a fast phase with a half-time of 0.6 microseconds that has been assigned to the reaction of bound cytochrome c2 [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137]. Modification of lysine-55 did not affect the half-time of this phase but decreased the apparent binding constant by a factor of 2. The derivatives modified at lysines-10, -88, -95, -97, -99, -105, and -106 surrounding the heme crevice did not show any detectable fast phase but only slow second-order phases due to the reaction of solution cytochrome c2. These lysines thus appear to be involved in binding cytochrome c2 to the reaction center in an optimal orientation for electron transfer. The involvement of lysines-95 and -97 is especially significant, since they are located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c. The reactions of horse cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or [(trifluoromethyl)phenyl]carbamoyl were also studied. The derivatives modified at lysines-22, -55, -88, and -99 far removed from the heme crevice had nearly the same half-times for the fast phase as native cytochrome c, 6 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings

We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N(epsilon2) coordination bond, and protonation of the N(delta1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N(epsilon2) position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N(epsilon1) position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N(delta1) atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N(epsilon2) atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.     

Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12

Summary A 37 kb fragment of DNA from an F-prime factor, F100-12, which showed a gene dosage effect on b-type cytochromes, was cloned with a cosmid vector, pHC79. Gel filtration of cytochromes and product analysis of the hybrid plasmids indicated that this fragment contained cybB, the structural gene for cytochrome b561. A chromosomal DNA fragment carrying the cybB gene was cloned by the plaque hybridization technique with Charon 4A as a vector. The gene was subcloned into pBR322 and was located in a 1.3 kb DNA fragment. It was concluded that the cybB gene is located on the chromosome of Escherichia coli K12.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - NADH reduced form of nicotinamide adenine dinucleotide