Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon

Abstract Membranes of the extremely thermoacidophilic archaeon Desulfurolobus ambivalens grown under aerobic conditions contain a quinol oxidase of the cytochrome aa ₃-type as the most prominent hemoprotein. The partially purified enzyme consists of three polypeptide subunits with apparent molecular masses of 40, 27 and 20 kDa and contains two heme A molecules and one copper atom. CO difference spectra suggest one heme to be a heme a ₃-centre. The EPR spectra indicate the presence of a low-spin and a high-spin heme species. Redox titrations of the solubilized enzyme show the presence of two reduction processes, with apparent potentials of + 235 and + 330 mV. The enzyme cannot oxidize reduced cytochrome c , but rather serves as an oxidase of caldariella quinone. Due to their very simple composition, D . ambivalens cell appear as a promising candidate to study Structure-function relationships of cytochrome aa ₃ in the integral membrane state.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration.

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

Synthesis of the Rhodopseudomonas viridis holo-cytochrome c2 in Paracoccus denitrificans

Abstract The gene encoding the Rhodopseudomonas viridis cytochrome c ₂ (cycA) has been introduced on a broad host range vector into Paracoccus denitrificans , leading to high-level expression of the holo-cytochrome with the heme moiety covalently attached to the apoprotein. The cytochrome was demonstrated to reside in the periplasmic space of the host cell. In contrast to R. viridis , aerobic rather than anaerobic growth conditions led to higher production levels of the holo-cytochrome in P. denitrificans . This heterologous expression system provides a suitable genetic background for the functional expression and mutagenesis of polypeptides involved in bacterial photosynthesis, offering the possibility of detailed structural and functional investigation.     

Plasmid-mediated virulence genes in non-typhoid Salmonella serovars

Abstract Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa ₃-type cytochrome c oxidase and the bo ₃-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb ₃-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc -type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.     

Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7

Abstract The role of cytochrome b ₅₆₂, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b ₅₆₂ did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form ( b ₅₆₂) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b ₅₆₂ was upstream of both the ' g _y= 1.89' Rieske FeS cluster and the a -type cytochromes. These results show that the b -type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.     

Copper effect on cytochrome b of photosystem II under photoinhibitory conditions

Toxic Cu (II) effect on cytochrome b ₅₅₉ under aerobic photoinhibitory conditions was examined in two different photosystem II (PSII) membrane preparations active in oxygen evolution. The preparations differ in the content of cytochrome b ₅₅₉ redox potential forms. Difference absorption spectra showed that the presence of Cu (II) induced the oxidation of the high-potential form of cytochrome b ₅₅₉ in the dark. Addition of hydroquinone reduced the total oxidized high-potential form of cytochrome b ₅₅₉ present in Cu (II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of cytochrome b ₅₅₉ during photoinhibitory treatment showed slower kinetics of Cu (II) effect on cytochrome b ₅₅₉ in comparison with the rapid loss of oxygen evolution activity in the same conditions. This result indicates that cytochrome b ₅₅₉ is affected after PSII centres are photoinhibited. The high-potential form was more sensitive to toxic Cu (II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of cytochrome b ₅₅₉ was completely lost; however, the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. The results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of cytochrome b ₅₅₉, respectively.     

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

A periplasmic location for the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus

Abstract Evidence is presented to show that the thiosulphate-oxidising multi-enzyme system from Thiobacillus versutus has a periplasmic location, and that the oxygen-binding site of the cytochrome oxidase ( aa ₃) is on the inner surface of the membrane. A scheme for the mechanism of generation of a proton motive force during electron flow from thiosulphate to oxygen via cytochrome c and aa ₃ is proposed.     

The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

Paracoccus denitrificans CcmG is a periplasmic protein–disulphide oxidoreductase required for c- and aa3-type cytochrome biogenesis; evidence for a reductase role in vivo

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein–disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG–PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c -type cytochromes, but also loss of spectroscopically detectable cytochrome aa ₃, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa ₃ and cytochrome c oxidase activity, although it did not restore c -type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein–disulphide bonds in certain protein substrates including c -type cytochrome polypeptides and/or polypeptides involved in c -type cytochrome biogenesis.     

The cytochrome bd terminal oxidase of Azotobacter vinelandii: Low temperature photodissociation spectrophotometry reveals reactivity of cytochromes b595 and d with both carbon monoxide and oxygen

Abstract Cytochromes d and b ₅₉₅ were studied by low temperature photodissociation of CO-ligated Azotobacter vinelandii membranes. White light or He-Ne laser irradiation revealed 436 and 594–597 nm absorption bands to be due to Fe¹¹ cytochrome b ₅₉₅. Oxy-cytochrome d (648 nm) was formed when the CO adduct was photolysed in the presence of oxygen. This was followed by ligand recombination (presumably oxygen) to the high-spin cytochrome b ₅₉₅, with a distinctive shift to shorter wavelengths of the α-band of the cytochrome, and a decrease in the oxygenated form. All spectral changes were light-reversible. We demonstrate the light-reversible binding of CO to both cytochromes b ₅₉₅ and d , and suggest migration of oxygen from cytochrome d to cytochrome b ₅₉₅ at a haem-haem binuclear centre during the oxidase reaction.     

Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.     

Purification and characterization of membrane-bound CO-reactive hemoprotein from Tetrahymena pyriformis mitochondria

Abstract A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis . It showed absorption peaks at 615 and 455 nm in the reduced form and an α peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a ₆₂₀' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis , it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a ₆₂₀' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis .     

REDOX PROPERTIES OF CYTOCHROME OXIDASE AND VASCULAR REACTIVITY OF ASTROCYTOMAS AND NEUROBLASTOMAS IN VIVO

Abstract— Dual wavelength reflection spectrophotometry was used to determine steady state changes in the reduction-oxidation ratio of cytochrome c oxidase ( a,a ₃) and vascular reactivity accompanying progressive growth of cortical and subcutaneous astrocytomas and neuroblastomas in vivo. Blood volume responses indicate that vessels invading the tumors retain regulatory reactivity typical of the body region of implantation and do not acquire those typical of the tissue of origin of the neoplastic cells. In comparison with non neoplastic tissue, early stage tumor growth was associated with highly oxidized ratios of cytochrome a,a ₃. A transition to highly reduced cytochrome a,a ₃ occurred during late stage tumor development. Such differences from normal cerebral tissues reflect alterations in micro-circulation and respiratory chain function accompanying the dynamics of tumor growth, and could provide a basis for selective therapeutic measures.