Interaction between uranium and the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>3</Subscript> of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> strain G20

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.     

Periplasmic oxygen reduction by Desulfovibrio species

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.     

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.     

Comparative studies of polyhemic cytochromes c isolated from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans (Norway)

Cytochrome c3 (Mr 26,000) has been characterized in Desulfovibrio vulgaris (Hildenborough) and its properties compared with polyhemic cytochromes c isolated from the same organism and from D. desulfuricans (Norway). It can be described as an octaheme cytochrome c3 constituted of two identical subunits. Absorption spectrum is similar to cytochrome c3 (Mr 13,000) and individual redox potentials have an average value of -180 mV.3 The N terminal sequence is compared with an homologous cytochrome isolated from D. desulfuricans Norway.     

Kinetic studies of the electron exchange reaction between the octaheme cytochrome c3 (Mr 26000) and the hydrogenase from Desulfovibrio desulfuricans Norway.

The octaheme cytochrome c3 (Mr 26000) from Desulfovibrio desulfuricans Norway was studied using cyclic voltammetry at the pyrolytic graphite electrode. The kinetics of reduction of the octaheme cytochrome c3 (Mr 26000) from D. desulfuricans Norway by the Ni-Fe-Se hydrogenase purified from the same organism was investigated by an electrochemical method. From cyclic voltammetry experiments a value of 8.108M-1S-1 was obtained for the second order homogenous rate constant of the electron transfer between the two proteins. Results are compared with similar experiments performed on the electron exchange between the tetrahemic cytochrome c3 (Mr 13000) and hydrogenase.     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family.

BACKGROUND: Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS: Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS: This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.     

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.     

Cytochrome c(3) mutants of Desulfovibrio desulfuricans

To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.     

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes.     

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.     

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria.     

Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP complex--a membrane-bound redox complex involved in the sulfate respiratory pathway

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.     

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.     

Ferredoxin electron transfer site on cytochrome c3. Structural hypothesis of an intramolecular electron transfer pathway within a tetra-heme cytochrome.

To specify electron exchanges involving Desulfovibrio desulfuricans Norway tetra-heme cytochrome c3, the chemical modification of arginine 73 residue, was performed. Biochemical and biophysical studies have shown that the modified cytochrome retains its ability to both interact and act as an electron carrier with its redox partners, ferredoxin and hydrogenase. Moreover, the chemical modification effects on the cytochrome c3 1H NMR spectrum were similar to that induced by the presence of ferredoxin. This suggests that arginine 73 is localized on the cytochrome c3 ferredoxin interacting site. The identification of heme 4, the closest heme to arginine 73, as the ferredoxin interacting heme helps us to hypothesize about the role of the three other hemes in the molecule. A structural hypothesis for an intramolecular electron transfer pathway, involving hemes 4, 3 and 1, is proposed on the basis of the crystal structures of D. vulgaris Miyazaki and D. desulfuricans Norway cytochromes c3. The unique role of some structural features (alpha helix, aromatic residues) intervening between the heme groups, is proposed.     

Electrophoretic and Immunological Differences Between the Cytochrome c3 of Desulfovibrio desulfuricans and that of Desulfovibrio vulgaris

The cytochrome c(3) of Desulfovibrio desulfuricans and that of D. vulgaris were purified to homogeneity as judged by disc gel electrophoresis and by ultracentrifugation. Both cytochromes had an oxidation-reduction potential of -205 +/- 5 mv at pH 7.0 and showed characteristic absorption bands at 525 and 553 nm in the reduced state. The molecular weights of the two cytochromes (calculated from sedimentation and diffusion data) were similar, with values of 13,500 to 14,300 for D. desulfuricans and 13,800 to 14,700 for D. vulgaris. The two cytochromes differed in their electrophoretic properties on Geon and polyacrylamide gel electrophoresis and did not share a common precipitating antigenic determinant as judged by immunodiffusion data.     

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Identification of a periplasmic C-type cytochrome as electron donor to the plasma membrane-bound cytochrome oxidase of the cyanobacterium Nostoc Mac

Photoautotrophically grown cyanobacterium Nostoc sp. strain Mac (PCC 8009) released up to about 10 nmol of a c-type cytochrome per ml packed cells after treatment with EDTA under conditions that left the plasma membrane absolutely intact as judged from the absence of cytosolic proteins in the supernatant. Spectra of the ascorbate reduced cytochrome revealed peaks at 553, 522 and 416 nm. The protein was purified to an A-553/A-275 ratio of 0.8. Midpoint potential (at pH 7), isoelectric point and apparent molecular weight of the cytochrome were +0.35 V, 8.6, and around 10,500, respectively. The cytochrome proved to be an excellent electron donor to the aa3-type cytochrome oxidase in both plasma and thylakoid membranes isolated and purified from Nostoc Mac. Chemoheterotrophic growth of the cells increased the level of periplasmic cytochrome c up to 10-fold and cytochrome oxidase activity of plasma membranes up to 90-fold. The periplasmic cytochrome also transferred electrons to photosystem I in illuminated thylakoid membranes. We conclude that cyanobacteria contain a periplasmic c-type cytochrome presumably identical to so-called cytochrome c6 or c-553 which has long been known as a photosynthetic (i.e. thylakoid-associated) redox protein in these organisms, and which is capable of donating electrons (from the periplasmic space) to the cytochrome oxidase in the plasma membrane and (from the thylakoid lumen) to both P700 and cytochrome oxidase in the thylakoid membrane.