Membrane-bound cytochrome c is an alternative electron donor for cytochrome aa3 in Nitrobacter winogradskyi.

We purified membrane-bound cytochrome c-550 [cytochrome c-550(m)] to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The cytochrome showed peaks at 409 and 525 nm in the oxidized form and peaks at 416, 521, and 550 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 18,400 on the basis of protein and heme c contents and 18,600 by gel filtration. The N-terminal amino acid sequence of cytochrome c-550(m) was determined to be A-P-T-S-A-A-D-A-E-S-F-N-K-A-L-A-S-A-?-A-E-?-G-A-?-L-V-K-P. We previously purified soluble cytochrome c-550 cytochrome c-550(s)] from N. winogradskyi and determined its complete amino acid sequence (Y. Tanaka, Y. Fukumori, and T. Y. Yamanaka, Biochim. Biophys. Acta 707:14-20, 1982). Although the sequence of cytochrome c-550(m) was completely different from that of cytochrome c-550(s), ferrocytochrome c-550(m) was rapidly oxidized by the cytochrome c oxidase of the bacterium. Furthermore, the liposomes into which nitrite cytochrome c oxidoreductase, cytochrome c oxidase, and nitrite were incorporated showed nitrite oxidase activity in the presence of cytochrome c-550(m). These results suggest that cytochrome c-550(m) may be an alternative electron mediator between nitrite cytochrome c oxidoreductase and cytochrome c oxidase.     

Comparison of binding and functional properties of two extrinsic components,Cyt c550 and a 12 kDa protein,in cyanobacterial PSII with those in red algal PSII

Cyt c550 and 12 kDa protein are two extrinsic proteins of photosystem II (PSII) found in cyanobacteria and some eukaryotic algae. The binding patterns of these two extrinsic proteins are different between cyanobacterial (Thermosynechococcus vulcanus) and red algal (Cyanidium caldarium) PSIIs [Shen and Inoue (1993) Biochemistry 32: 1825; Enami et al. (1998) Biochemistry 39: 2787]. In order to elucidate the possible causes responsible for these differences, we first cloned the psbV gene encoding Cyt c550 from a red alga, Cyanidium caldarium, which was compared with the homologous sequences from other organisms. Cross-reconstitution experiments were then performed with different combinations of the extrinsic proteins and the cyanobacterial or red algal PSII. (1). Both the cyanobacterial and red algal Cyt c550 bound directly to the cyanobacterial PSII, whereas none of them bound directly to the red algal PSII, indicating that direct binding of Cyt c550 to PSII principally depends on the structure of PSII intrinsic proteins but not that of Cyt c550 itself. (2). Cyt c550 was functionally exchangeable between the red algal and the cyanobacterial PSII, and the red algal 12 kDa protein functionally bound to the cyanobacterial PSII, whereas the cyanobacterial 12 kDa protein did not bind to the red algal PSII. (3). The antibody against the cyanobacterial or red algal 12 kDa protein reacted with its original one but not with the homologous protein from the other organism, whereas the antibody against the red algal Cyt c550 reacted with both cyanobacterial and red algal Cyt c550. These results imply that the structure and function of Cyt c550 have been largely conserved, whereas those of the 12 kDa protein have been changed, in the two organisms studied here.     

Photosynthetic cytochrome c550

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl(-) and Ca(2+) ions to the oxygen evolving complex and protects the Mn(4)Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (E(m)) of the purified soluble form (from -250 to -314mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an E(m) value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22?) between cyt c550 and the nearest redox cofactor (Mn(4)Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

The structure of Paracoccus denitrificans cytochrome c550.

The crystal structure of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been solved by x-ray diffraction to a resolution of 2.45 A. In both amino acid sequence and molecular structure it is evolutionarily homologous with mitochondrial cytochrome c from eukaryotes and photosynthetic cytochrome c2 from purple non-sulfur bacteria. All of these cytochromes c have the same basic folding pattern, with surface insertions of extra amino acids in c550. Various strains of c2 have all, some, or none of the extra insertions observed in c550. The hydrophobic heme environment, position of aromatic rings, and structure and environment of the heme crevice, are virtually identical in cytochromes c55o, c, and c2. Radical changes observed at all regions on the molecular surface except the heme crevice argue for the importance of the crevice and the exposed edge of the heme in the transfer of electrons to and from the cytochrome molecule.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

A high redox potential form of cytochrome c550 in photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ～300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.     

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.     

Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus

Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential ( E'(m)) of +390 mV in about half of the centers and +275 mV in the other half. The high-potential form, whose E'(m)is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E'(m) of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E'(m)when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0). The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Redox potential of cytochrome c550 in the cyanobacterium Thermosynechococcus elongates

Cytochrome c550 (cyt c550) from photosystem II (PSII) exists in the PSII-bound form but can be released from PSII by treatment with divalent cations or Tris, yielding the isolated form. We calculated heme redox potentials (Em) based on the crystal structures of cyt c550 by solving the Poisson-Boltzmann equation. In the isolated form, the calculated Em are -240 mV at pH 6.0 and -352 mV at pH 9.0. This pH-dependence is predominantly due to deprotonation of the heme-propionic group near Asn-49. In the PSII-bound form, the calculated E(m) was up-shifted by 160 mV versus the isolated form due to a conformational change of protein backbone, yielding Em=-84 mV.     

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.     

Isolation of water-soluble cytochromes from cyanobacteria by adsorption chromatography

A simple procedure using ammonium sulfate to fractionate water-soluble cytochromes c553 and c550 on Sephacryl S-200 gel is described. The usefulness of this procedure has been studied using the crude extracts of mesophilic cyanobacteria. It was found that almost all the cytochromes were adsorbed on to the gel at 2.34 M ammonium sulfate and were eluted at decreasing salt concentrations. The cytochromes were free of interfering phycobiliproteins and thus were suitable for the study of isoelectric points. It was also found that this procedure allowed a clear separation of the cytochromes based on their hydrophobicities. The order of elution was cytochrome c553, then cytochrome c550, indicating that c550 is more hydrophobic than c553. All these results show that this procedure provides both a simplified and an efficient purification of the cytochromes and insight into their surface properties. The cytochromes of Microcystis aeruginosa were purified to homogeneity using this procedure and other existing ones. Homogeneous cytochromes c553 and c550 were chromatographed on Sephacryl S-200 at 1.75 M (NH4)2SO4 and found to elute in the same order as reported earlier for the cytochromes in the crude extracts. In addition, cytochrome c550 was found to be more heat resistant and less water soluble than cytochrome c553.     

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

The amino acid sequence of cytochrome c553 from Microcystis aeruginosa

Cytochrome c553 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aeruginosa and determined its amino acid sequence. When the amino acid sequence of this protein is compared to sequences of cytochromes c553 from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria. Cyanobacteria and algae also contain cytochrome c550 (Mr 15,500) which is quite different from cytochrome c553 (Mr 10,500). When the amino acid sequence of cytochrome c553 is compared to that of cytochrome c550, two regions of similar sequence are recognized.     

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: study of redox mutants

Cytochrome c(550) is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c(550) we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and -30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c(550) is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c(550) was also constructed. It showed a lowered affinity for Cl(-) and Ca(2+) as reported earlier for the cytochrome c(550)-less Synechocystis 6803 mutant, but it showed a shorter lived S(2)Q(B)(-) state, rather than a stabilized S(2) state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c(550).     

Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis

Bacillus subtilis expresses a cytochrome c-550nm that participates in respiratory electron transfer and is an integral membrane protein. Analysis of the B. subtilis cytochrome c-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990) J. Biol. Chem. 265, 13939-13948]. We have purified cytochrome c-550nm from wild-type B. subtilis and B. subtilis transformed with the shuttle vector pHP13 containing the gene for B. subtilis cytochrome c-550nm (cccA). In B. subtilis transformed with pHP13/cccA there is better than eightfold more membrane-bound cytochrome c-550nm than in wild-type B. subtilis. The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six-histidine tag has been added to the C-terminus of cytochrome c-550nm from B. subtilis as a further aid for purification. This strain produces cytochrome c-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography. UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines. Neither form of B. subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type and his-tagged, of cytochromes c have similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates for B. subtilis cytochrome c oxidase. We conclude that the addition of the histidine tag eases the purification of cytochrome c-550nm from B. subtilis plasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein.     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.     

Amino acid sequence of Paracoccus denitrificans cytochrome c550.

The amino acid sequence of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been established by a combination of standard chemical techniques and interpretation of a 2.5 A resolution x-ray electron density map. Peptides derived from a trypsin digest were chemically sequenced, and then ordered by fitting them to the density map. The amino acid compositions of chymotryptic peptides confirmed the x-ray map ordering the tryptic peptides. The amino acid sequence of this respiratory, prokaryotic cytochrome with 134 residues is discussed in relation to those of eukaryotic respiratory cytochrome c (103 to 113 amino acids), and prokaryotic, photosynthetic c2 (103 to 124 amino acids). At the primary structure level, c and c550 differ no more from cytochromes c2 than the various cytochromes c2 do from one another. It is suggested that the respiratory electron transport chain in prokaryotes and eukaryotes is a relatively late evolutionary offshoot of the photosynthetic electron transport chain in purple non-sulfur bacteria.