b-Type cytochromes in plasma membranes ofPhaseolus vulgaris hypocotyls,Arabidopsis thaliana leaves, andZea mays roots

Summary The plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb ₅₆₁ (cytb ₅₆₁), has its characteristic -band absorbance close to 561 nm wavelength at room temperature. Cytb ₅₆₁ was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb ₅₆₁ did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb ₅₆₁ was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb ₅₆₁ bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb ₅₆₁ from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb ₅₆₁ from different sources.Abbreviations cyt b ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - PAGE polyacrylamide gel electrophoresis     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Higher-plant plasma membrane cytochromeb 561: A protein in search of a function

Summary During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb ₅₆₁ (cytb ₅₆₁) according to the wavelength maximum of its -band in the reduced form. More recent evidence suggests that this protein is homologous to ab-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cytb ₅₆₁ is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromesb ₅₆₁ probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromesb ₅₆₁. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cytb ₅₆₁ is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb ₅₆₁, compares it to the animal cytb ₅₆₁, and discusses the possible physiological function of these proteins in plants.Abbreviations Asc ascorbate - cyt cytochrome - DHA dehydroascorbate - E₀ standard redox potential - EST expressed sequence tag - His histidine - MDA monodehydroascorbate - Met methionine - PM plasma membrane     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Cytochrome b561 is not fatty acylated but acetylated at amino terminus in chromaffin vesicle membranes: an approach for the identification of posttranslational modification of transmembrane proteins

Summary.  We examined the nature of the posttranslational modification of bovine cytochrome b ₅₆₁, a membrane-spanning protein and an essential component of neuroendocrine secretory vesicles. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed two populations in the partially digested fragments of cytochrome b ₅₆₁, which were obtained by controlled treatment of cytochrome b ₅₆₁-proteoliposomes with trypsin. One population, containing the posttranslationally modified amino-terminal region, showed molecular masses which were by about 40 Da larger than the theoretical molecular masses. The other population, without the modified amino-terminal region, showed a reasonable matching with the theoretical masses. This result suggested that the posttranslational modification occurred only in the amino-terminal region. The amino-terminal peptide was isolated by tryptic peptide mapping followed by treatment with acylamino-acid-releasing enzyme. Amino acid sequence and MALDI-TOF-MS analyses of the amino-terminal peptide showed that the initial Met residue was acetylated. There was no other posttranslational modification in the amino-terminal region, such as covalent fatty acylation through an ester linkage to Ser or Thr residues. Received May 9, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Molecular Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1-1, Nada-ku, Hyogo 657-8501, Japan.     

Structure prediction for the di-heme cytochrome b561 protein family

Summary.  Atomic models possessing the common structural features identified for the cytochrome b ₅₆₁ (cyt b ₅₆₁) protein family are presented. A detailed and extensive sequence analysis was performed in order to identify and characterize protein sequences in this family of transmembrane electron transport proteins. According to transmembrane helix predictions, all sequences contain 6 transmembrane helices of which 2–6 are located closely in the same regions of the 26 sequences in the alignment. A mammalian (Homo sapiens) and a plant (Arabidopsis thaliana) sequence were selected to build 3-dimensional structures at atomic detail using molecular modeling tools. The main structural constraints included the 2 pairs of heme-ligating His residues that are fully conserved in the family and the lipid-facing sides of the helices, which were also very well conserved. The current paper proposes 3-dimensional structures which to our knowledge are the first ones for any protein in the cyt b ₅₆₁ family. The highly conserved His residues anchoring the two hemes on the cytoplasmic side and noncytoplasmic side of the membrane are in all proteins located in the transmembrane helices 2, 4 and 3, 5, respectively. Several highly conserved amino acids with aromatic side chain are identified between the two heme ligation sites. These residues may constitute a putative transmembrane electron transport pathway. The present study demonstrates that the structural features in the cyt b ₅₆₁ family are well conserved at both the sequence and the protein level. The central 4-helix core represents a transmembrane electron transfer architecture that is highly conserved in eukaryotic species. Received May 12, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, Biological Research Centre, P.O. Box 521, 6701 Szeged, Hungary. E-mail: tpali@nucleus.szbk.u-szeged.hu     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

<Emphasis Type="Italic">Haloarcula marismortui</Emphasis> cytochrome <Emphasis Type="Italic">b-</Emphasis>561 is encoded by the <Emphasis Type="Italic">narC</Emphasis> gene in the dissimilatory nitrate reductase operon

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was −27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.     

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with E _m,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (E _m,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c ₂ class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c. Received: 8 June 1998 / Accepted: 25 August 1998     

Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein

Tumor suppressor protein 101F6, a gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, has recently been identified as a member of the cytochrome b561 (Cyt-b561) protein family by sequence homology. The His₆-tagged recombinant mouse tumor suppressor Cyt-b561 protein (TSCytb) was recently expressed in yeast and purified, and the ascorbate reducibility was determined. TSCytb is auto-oxidizable and has two distinct heme b centers with redox potentials of ~40 and ~140 mV. Its split α-band in the dithionite-reduced spectrum at both 295 and 77 K is well resolved, and the separation between the two α-peaks is ~7 nm (~222 cm⁻¹). Singular value decomposition analysis of the split α-band in the ascorbate-reduced spectra revealed the presence of two major spectral components, each of them with split α-band but with different peak separations (6 and 8 nm). Similar minor differences in peak separation were obtained when the split α-bands in ascorbate-reduced difference spectra at low (<1 mM) and high (>10 mM) ascorbate concentrations were analysed. According to low-temperature electron paramagnetic resonance (EPR) spectroscopy, the two heme b centers are in the low-spin ferric state with maximum principal g values of 3.61 and 2.96, respectively. These values differ from the ones observed for other members of the Cyt-b561 family. According to resonance Raman spectroscopy, the porphyrin rings are in a relaxed state. The spectroscopic results are only partially in agreement with those obtained earlier for the native chromaffin granule Cyt-b561.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with E _m,7 of +180, +72 and –5 mV (561–575 nm), and +244 and +27 mV (551–540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with E _m,7 of +60 and –45 mV and +290, +250, +135, –20 and –105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (E _m,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c ₂, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85–90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa ₃ type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c ₂ or a branched respiratory chain. Received: 25 February 1997 / Accepted: 20 May 1997     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria

Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 μM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c ₃(M _r. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c ₃ from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c ₇ from Desulfuromonas acetoxidans. Results with cytochromes c ₃ and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate. Received: 14 April 2000 / Received revision: 6 July 2000 / Accepted: 9 July 2000     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997     

Energy and Electron Transfer Systems of Chlamydomonas reinhardi. I. Photosynthetic and Respiratory Cytochrome Systems of the Pale Green Mutant

The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b₅₆₃ and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b₅₅₉ is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

The NADH-dependent Fe3+-chelate reductases of tomato roots

The NADH-dependent Fe³⁺-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe³⁺-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe³⁺-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol · min⁻¹ · mg⁻¹) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol · min⁻¹ · mg⁻¹). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497–1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b ₅ reductase flavoprotein group. Received: 6 November 1996 / Accepted: 21 January 1997     

Exploration of Respiratory Chain of <Emphasis Type="Italic">Nocardia asteroides:</Emphasis> Purification of Succinate Quinone Oxidoreductase

Nocardia asteroides is a pathogenic bacterium that causes severe pulmonary infections and plays a vital role in HIV development. Its electron transport chain containing cytochromes as electron carriers is still undiscovered. Information regarding cytochromes is important during drug synthesis based on cytochrome inhibitions. In this study we explored the electron transport of N. asteroides. Spectroscopic analysis of cytoplasm and membranes isolated from N. asteroides indicates the presence of soluble cytochrome-c, complex-II and the modified a ₁ c ₁ complex as the terminal oxidase. The molecular weight of the respiratory complex-II isolated and purified from the given bacterium was 103 kDa and was composed of three subunits, of 14, 26 and 63 kDa. Complex-II showed symmetrical α-absorption peaks at 561 nm in the reduced state. Spectral analysis revealed the presence of only one heme b molecule (14-kDa subunit) in complex-II, which was confirmed by heme staining. Heme b content was found to be 9.5 nmol/mg in complex-II. The electron transport chain of N. asteroides showed the presence of soluble cytochrome-c, cytochrome-a ₁ c ₁ and cytochrome-b.