Algal Cytochrome Research Articles

Crystallization and Structural Analysis of Cytochromec6from the DiatomPhaeodactylum tricornutumat 1.5 Å Resolution

We determined for the first time the crystal structure of diatom cytochrome c 6 from Phaeodactylum tricornutum at 1.5 Å resolution. The overall structure of the protein was classified as a class I c-type cytochrome. The physicochemical properties of the protein were examined by denaturation with guanidine hydrochloride and urea, and compared with those of other algal cytochrome c 6.

Cytochrome c6A: discovery, structure and properties responsible for its low haem redox potential

Cytochrome c(6A) is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c(6), but is unable to fulfil the same function of transferring electrons from cytochrome f to Photosystem I. A key feature of cytochrome c(6A) is that its haem midpoint potential is more than 200 mV below that of cytochrome c(6) (E(m) approximately +340 mV) despite both cytochromes having histidine and methionine residues as axial haem-iron ligands. One salient difference between the haem pockets is that a valine residue in cytochrome c(6A) replaces a highly conserved glutamine residue in cytochrome c(6). This difference has been probed using site-directed mutagenesis, X-ray crystallography and protein film voltammetry studies. It has been found that the stereochemistry of the glutamine residue within the haem pocket has a destabilizing effect and is responsible for tuning the haem's midpoint potential by over 100 mV. This large effect may have contributed to the evolution of a new biological function for cytochrome c(6A).

Cloning, expression and purification of cytochrome c(6) from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure.

The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

Modulation of Heme Redox Potential in the Cytochrome c6 Family

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.

Expression of the Algal Cytochrome c6 Gene in Arabidopsis Enhances Photosynthesis and Growth

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.

Cross-reconstitution of extrinsic cytochrome c-550 and 12 kDa protein between cyanobacterial and red algal PSII

Red algal and cyanobacterial PSII contain two similar extrinsic proteins, namely, cytochrome c-550 and a 12 kDa protein. Their amino acid sequences showed a relatively high homology of about 40% between red algae and cyanobacteria. In this study, cross-reconstitution experiments of the extrinsic proteins with red algal (Cyanidium caldarium) or cyanobacterial (Synechococcus vulcanus) PSII were performed in order to compare the binding and functional properties of the extrinsic proteins between red algae and cyanobacteria. Red algal cytochrome c-550, as well as cyanobacterial cytochrome c-550, bound directly to cyanobacterial PSII. However, none of them bound directly to red algal PSII. This indicates that direct binding of cytochrome c-550 to PSII depends upon the structure of intrinsic proteins of PSII but not cytochrome c-550 itself. Red algal 12 kDa protein functionally bound to cyanobacterial PSII, while cyanobacterial 12 kDa protein did not bind to red algal PSII. The antibodies raised against cyanobacterial or red alga 12 kDa protein did not cross-react with red algal or cyanobacterial 12 kDa protein, respectively. These results imply that, while the structure and function of cytochrome c-550 were rather conserved, those of the 12 kDa protein have been changed during evolution from cyanobacteria to red algae.

Characterization and amino acid sequences of cytochromes c6 from two strains of the green alga Chlorella vulgaris.

Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of +362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of +355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif -CXXCH-, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45%.

Mitochondrial genetics of Chlamydomonas reinhardtii: resistance mutations marking the cytochrome b gene.

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.

Kinetics of reduction by free flavin semiquinones of algal cytochromes and plastocyanin

It had been shown that plastocyanin and cytochrome c-553 are functionally interchangeable in algae and that the physiological electron transfer reactions are sensitive to ionic strength. The isoelectric points of these proteins range from very acidic to basic depending upon species, and naturally occurring amino acid substitutions of charged residues have been shown to affect the kinetics of electron transfer, presumably through alteration of protein net charge. We have now shown that these naturally occurring amino acid substitutions also affect the kinetics of nonphysiological electron transfer reactions, and that we can quantitate the extent of nonconservation of charge. The reduction of plant and algal proteins by FMN semiquinone is sensitive to ionic strength and the effects can be correlated with net protein charge with regard to sign, but not to magnitude, with the charge at the site of electron transfer varying from +3 through 0 to −3. We had previously observed in a large variety of electron transfer proteins from bacteria ( G. Tollin, T. E. Meyer, and M. A. Cusanovich (1986) Biochim. Biophys. Acta 853, 29–41 ) that charge localized at the site of electron transfer, rather than net protein charge, was more likely to affect kinetics. This also appears to be the case with the algal proteins. By comparison of protein structures, we have been able to predict which substitutions are likely to be responsible for the kinetic effects in the algal proteins and to discuss the implications of such changes for function.

Oxyfluorfen and Lipid Peroxidation: Protein Damage as a Phytotoxic Consequence

Protein damage, as a primary phytotoxic consequence of in vivo lipid peroxidation, induced by the diphenyl ether herbicide oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] at a concentration of 10 μM, was measured with the green algaScenedesmus acutus. In the light, water-soluble proteins are destroyed by a herbicide-induced peroxidation process that can be detected by production of fluorescent products and loss of specific amino acid residues of proteins. The water-soluble cytochrome c-553 and the membrane-bound cytochrome f-553, components of the photosynthetic electron transport, were specifically used as sensitive markers for protein damage, measured as decrease of redox reactions of the cytochromes. Under peroxidizing conditions, destruction of the algal cytochrome c is significantly higher than destruction of membrane-bound components, such as cytochrome f and chlorophyll. Protection against protein loss is achieved by the nonbiological antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) or the photosynthesis inhibitor diuron [N′-(3,4-dichlorophenyl)-N,N-dimethylurea].

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c 2 and c′ have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c′ found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c′ and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c 1 and chloroplast cytochrome f.

Studies on algal cytochromes. V. Purification and characterization of cytochrome c-552 from a red alga, Polysiphonia urceolata.

Cytochrome c-552 was extracted from a red alga, Polysiphonia urceolata, by immersion of the frozen trichomes in deionized water. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography, hydroxyapatite column chromatography, and Bio-Gel P-10 gel filtration. The ferrocytochrome c-552 has absorption maxima at 551.5(alpha), 522.5(beta), 416.3(gamma), 318(delta), 292, and 270 nm; those of the ferricytochrome are at 525, 408.5(gamma), and 358 nm. The pyridine ferrohemochrome showed absorption maxima at 550(alpha), 520(beta), and 414 nm(gamma). The alpha-band of the ferrocytochrome is symmetric without any shoulder at room temperature, and does not split even at liquid nitrogen temperature. The ferricytochrome showed a weak absorption shoulder at 695 nm, suggesting a methionine sulfur to be the sixth ligand of heme c iron. The cytochrome is oxidized by ferricyanide and reduced by ferrocyanide, cysteine, ascorbate, and hydrosulfite. Autoxidation was not observed. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferricyanide system to be 0.333 volt at pH 7.0 and 25 degrees C. The isoelectric points of the ferro- and ferricytochromes were determined to be at pH 3.85 and 4.02, respectively, by the isoelectric focusing method. The molecular weight was estimated to be about 12,000 from the results of gel filtration and SDS polyacrylamide gel electrophoresis.

Studies on algal cytochromes II. Some physical and chemical properties of further purified Petalonia cytochrome c-553.

A cytochrome c-553 was isolated from a brown alga, Petalonia fascia, and its physical and chemical properties were investigated. At liquid nitrogen temperature, the α-, β- and γ-band of this cytochrome shifted 1-2 nm to the shorter wavelength in comparison with those at room temperature. The α-band split into two peaks, 551.5 (major band) and 546.5 nm (minor band), at the low temperature. The cytochrome in high concentration showed a shoulder at 695 nm in its oxidized state, suggesting a methionine residue to be the sixth ligand of heme iron. The molecular weight was estimated to be about 10,000 containing one mole of heme c based on analyses of gel filtration, sodium dodecyl sulfate acrylamide gel electrophoresis, amino acid composition and iron content. The isoelectric points of the ferro- and ferricytochromes were estimated to be at pH 4.1 and 4.3, respectively, by the isoelectric focusing method. The amino acid composition of this cytochrome was Lys6, Arg1, Asp15, Thr5, Ser6, Glu11, Pro2, Gly7, Ala7, Cys2, Val6, Met3, Ile6, Leu3, Tyr1, Phe3, Trp1, with a total of 86 residues. The amino- and carboxylterminal sequences and four chymotryptic peptide sequences were compared with those of other cytochromes c to show that these cytochromes were homologous to one another.

Isolation and Some Molecular Properties of Plastidic Algal Cytochrome b-559

Abstract Cytochrome b-559, Algae, Spinach Cytochrome b-559, an integral protein of diloroplast thylakoids, was prepared in a homogeneous form from the alga Bumilleriopsis filiformis. The protein is easily denatured and can be solubilized by treatment of isolated thylakoids with high concentrations of urea and detergents in addition to weak sonification. The reduced form exhibits absorption maxima at 559, 530 and 429 nm. By comparative determination, a molecular weight of 17 000 was found for the protein from Bumilleriopsis, whereas that for spinach has 37 000 daltons. Both proteins have a low, but variable lipid content which is not a necessary part of the enzymatically active cytochrome b-559. The purified cytochrome exhibits a low midpoint the preparation a transient “high-potential” form (reducible by hydroquinone) was also solubilized.

Resonance raman studies of a c type algal cytochrome deuterium shifts and a comparison with mammalian cytochrome c

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H 2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment of the vibrations of the c type heme in the algal cytochrome.

The cytochrome fold and the evolution of bacterial energy metabolism

Respiratory cytochrome c from eukaryotes, photosynthetic c2 from purple nonsulfur bacteria, and respiratory c550 from Paracoccus (formerly Micrococcus) denitrificans, have been shown by X-ray structure analysis and amino acid sequence comparisons to form a unified subfamily of evolutionarily homologous proteins (cytochromes c2) in which the differences between respiratory and photosynthetic cytochromes are no greater than the variation among photosynthetic cytochromes c2. From sequence and structure considerations alone, these cytochromes are indistinguishable as to origin or function. Low resolution (4 Å) X-ray analysis of Pseudomonas cytochrome c551 shows it to have the same overall folding pattern as cytochrome c, with a massive deletion at the bottom of the molecule. This information enables amino acid sequence homologies to be extended with reasonable probability of correctness to all cytochromes c551, to Chlorobium c555, Pseudomonas c5, algal cytochromes f, and even c553 from Desulfovibrio. All of these proteins appear to comprise a broader family of evolutionarily related electron carriers. It is proposed that bacterial and eukaryotic oxygen respiration arose from the dual-function photosynthetic and respiratory electron chain in purple non-sulfur bacteria, by the loss of photosynthetic capabilities. A detailed outline for the evolution of photosynthesis and respiration in bacteria is presented, involving first a symbiotic sulfur cycle, then the development of the Krebs cycle machinery and finally the evolution of the present-day symbiotic oxygen cycle between plants and animals.

The amino acid sequence of cytochrome f from the brown alga Alaria esculenta (L.) Grev.

Cytochrome f was isolated from the brown alga Alaria esculenta and the amino acid sequence was determined. The native haemoprotein has a molecular weight of 9800 and consists of a single polypeptide chain of 86 amino acid residues with a haem group bonded to cysteine residues at positions 14 and 17. The N-terminus is not acetylated and no methylated lysines were found. Sequences of three other algal cytochromes f were compared with that of Alaria and 22 out of 92 positions were common to the four sequences. One-half of these conserved sites occur between positions 49 and 63. Detailed evidence for the amino acid sequence of Alaria cytochrome has been deposited as Supplementary Publication SUP 50048 (6 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10 7 M −1 · s −1, at 298 °K and pH 7.0, with activation parameters ΔH ‡ = 44 kJ · mole −1 and ΔS ‡ = +46 J · mole −1 · ° K −1 . Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10 5, 7.5 · 10 5 and 1.0 · 10 6 M −1 · s −1, respectively. Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10 6 and approx. 2 · 10 7 M −1 · s −1 for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively. Rate constants of 115 and 80 M −1 · s −1 were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10 4 and 8 · 10 4 M −1 · s −1. The results are discussed in relation to photosynthetic electron transport.

Enzymatic reduction of algal cytochrome f

Summary The purified f-type cytochrome 553 of the alga Bumilleriopsis filiformis Vischer (Xanthophyceae) is used as electron acceptor (from NADPH) in a diaphorase system with ferredoxin-NADP reductase (E.G. 1.6.7.1; 1.6.1.1) and with and without ferredoxin of the alga present in the assay. Some enzymatic criteria of the system and of the algal cytochrome 553 in particular are thereby described. The latter is compared in this respect with mammalian cytochrome c: a) There is a substantial reactivity of cytochrome 553 with the reductase, ferredoxin not being necessary. b) Ferredoxin stimulates reduction about 10 times in case of cytochrome 553 and 80 to 90 times with mammalian cytochrome c at a common pH optimum of about 7. The Km is 0.5 μM ferredoxin with cytochrome 553 used as the terminal electron acceptor and 1.72 μM ferredoxin with cytochrome c. c) The superoxide radical is important only in cytochrome c reduction without ferredoxin present. Ferredoxin abolishes the effect of oxygen during cytochrome reduction. d) Furthermore, details of substitution of ferredoxin by potassium ferrocyanide are given and the influence of reduced thionicotinamide-NADP on cytochrome reduction.

Enzymic redox reactions of cytochromes c

The two cytochromes c for which detailed tertiary structures have been obtained recently—cytochrome c from mammalian mitochondria and cytochrome c 2 from Rhodospirillum rubrum—have been compared in reactivity in the physiologic redox systems for which the former is the natural substrate. These systems are the mitochondrial DPNH-cytochrome c reductase (complex I–III) and cytochrome c oxidase (complex IV). In addition, the effects of antimycin A (a specific inhibitor in the reductase system) and L-polylysine (usually employed to inhibit the oxidase system) on the reduction and oxidation of cytochromes c and c 2 have been studied. Preliminary data for these two cytochromes, as well as for five others representative of different classes of bacterial and algal cytochrome c are presented. Implications of these findings for current attempts to rationalize the structural features of cytochromes c are considered.