The amino acid sequence of Nitrosomonas europaea cytochrome c-552

The complete amino acid sequence of cytochrome c-552 derived from the chemoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea was determined. The cytochrome consisted of 81 amino acid residues, and its molecular weight was calculated to be 9098 including heme c. Although the sequence of cytochrome c-552 was highly homologous to those of cytochromes c-551, which were known as the electron-donating components to dissimilatory nitrite reductase in pseudomonads, cytochrome c-552 differed from cytochrome c-551 in two points: (1) the sequence of cytochrome c-552 was shorter by two amino acid residues than that of cytochrome c-551 at the N-terminus and (2) one amino acid insertion was present in cytochrome c-552.     

N-terminal amino acid sequence of cytochrome c-552 from Nitrosomonas europaea

Nitrosomonas europaea is an ammonia-oxidizing bacterium which contains multiple c-type cytochromes. Few of these components have been assigned physiological roles, but on the basis of molecular weight and redox potential cytochrome c-552 has been considered to be an analogue of the mitochondrial cytochrome-c family of proteins. We present the N-terminal amino acid sequence (47 residues) of cytochrome c-552 and show that this protein is most closely related to the group of small cytochrome-c components from pseudomonads (cytochromes c-551) and is probably evolutionarily distant from the analagous protein (cytochrome c-550) from the nitrite-oxidizing bacterium Nitrobacter agilis.     

The reaction of Euglena gracilis cytochrome c-552 with nonphysio-logical oxidants and reductants.

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c₂. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c₂.     

1H NMR studies of the heme iron coordination in cytochrome c-552 from Euglena gracilis.

The coordination of the heme iron in cytochrome c-552 from Euglena gracilis was investigated by 1H NMR studies at 360 MHz. The data imply that the axial heme ligands are His-14 and Met-56 in both the oxidized and the reduced protein. Studies of mixed solutions of ferro- and ferricytochrome c-552, which provided much of the information on the heme structure, also showed that the intermolecular electron exchange is characterized by a bimolecular rate constant of 5-10(6) mol-1-s-1 at 29 degrees C, which is three orders of magnitude faster than the corresponding reaction in solutions of mammalian cytochromes c.     

Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8

The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented. The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides. The sequence is homologous to c-type cytochromes, especially in the heme-binding region.     

1H NMR studies at 360 MHz of the aromatic amino acid residues in ferrocytochrome c-552 from Euglena gracilis.

The resonances of the aromatic rings in the 1H NMR spectra at 360 MHz of ferrocytochrome c-552 of Euglena gracilis were investigated by double resonance techniques. The spin systems of the two tryptophan and four of the tyrosine residues could be identified. This analysis of the aromatic region of the 1H NMR spectrum provided evidence that His-14 is bound to the heme iron. It gave also some insight into the molecular dynamics of ferrocytochrome c-552 in that it showed that of the six aromatic rings, four tyrosines were rotating rapidly about the Cbeta-Cgamma bond, while one tyrosine and the single phenylalanine were restricted in their rotational mobilities by their environmnent in the protein.     

1H-NMR studies of structural homologies between the heme environments in horse cytochrome c and in cytochrome c-552 from Euglena gracilis

With the use of proton-proton Overhauser enhancement experiment the spatial arrangement relative to the heme group of amino acid side chains in the heme crevice of horse ferrocytochrome c and ferrocytochrome c-552 from euglena gracilis was investigated. From these data and the known crystal structure for mammalian cytochromes c, individual assignments were obtained for several aromatic residues in horse ferrocytochrome c. This then provided a basis for delineating homologies between the polypeptide conformations near the heme group in horse ferrocytochrome c and ferrocytochrome c-552, for which no crystal structure has as yet been described.     

The redox potential of cytochrome c-552 from Euglena gracillis: a thermodynamic study.

The redox potentials for cytochrome c-552 at different ionic strengths, pH 7, have been determined, together with the thermodynamic parameters of the redox reaction. The effects of the electrostatic media on the redox potential of cytochrome c-552 do not depend on the nature of the ions employed. At 25 °C and pH 7 the observed potentials depend on the ionic strength, I, according to the equation: $\text{E}{}_{\mathrm{obs}}{}^{\mathrm{o}}\text{= 0.280 + .525 (I}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(I + I}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{))}$. The significance of the ionic strength dependence of the redox potentials and their derived thermodynamic parameters are discussed and compared to those of mammalian cytochrome c. It is concluded that the redox potentials for ionic strength approaching zero are not affected by the overall net charge of the proteins; at finite ionic strengths, the protein charges play a very important role in determining the observed redox potentials.     

The complete amino acid sequence of Nitrobacter agilis cytochrome c-550

The amino acid sequence of cytochrome c-550 from the chemoautotroph, Nitrobacter agilis, was completed by using solid-phase sequencing and conventional procedures. The cytochrome was composed of 109 amino acid residues and its molecular weight was calculated to be 12375 including haem c. The cytochrome was homologous to eukaryotic cytochromes c and some photosynthetic bacterial cytochromes c2. In particular, its primary structure was very similar to that of Rhodopseudomonas viridis cytochrome c2. Some of its properties were compared with those of other cytochromes c on the basis of the primary structure.     

The role of lysines in Euglena cytochrome c-552. Chemical modification studies.

2-Hydroxy(p-hydroxyphenyl)-acetaldoxime, the alternative precursor to p-hydroxyphenylacetonitrile in dhurrin biosynthesis, was synthesized and its effectiveness as a substrate was examined in a microsomal enzyme system from sorghum seedlings. The hydroxyaldoxime was slowly converted to p-hydroxymandelonitrile when compared with p-hydroxyphenylacetonitrile and p-hydroxyphenylacetaldoxime. Moreover, radioactivity from [U-¹⁴C]tyrosine was efficiently incorporated by trapping experiments into both the nitrile and aldoxime, but not into the hydroxyaldoxime. The reaction products formed on hydroxylation of the nitrile by the microsomal enzyme were identified as p-hydroxybenzaldehyde, HCN, and H₂O. Under anaerobic conditions, the nitrile was produced from the aldoxime and accumulated without undergoing hydroxylation. These results establish p-hydroxyphenylacetonitrile and not 2-hydroxy(p-hydroxyphenyl)-acetaldoxime as the intermediate in the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis.     

The amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185

The evidence for the amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185 is reported. The sequence was determined by manual Edman degradation of tryptic and chymotryptic peptides using the DABITC/PITC double-coupling method; some peptides were further cleaved by partial acid hydrolysis and with Staphylococcus aureus protease. The sequence overlaps 13-15, 83-85 and 106-108 as well as the region 113-118 involving the haem-binding sequence Cys-Xaa-Xaa-Cys-His were deduced by homology with cytochrome c-556 from Agrobacterium tumefaciens strain B2a. The identity of histidine at position 6 has been inferred from fast-atom bombardment experiments on the N-terminal tryptic peptide, and Asp-63 was deduced from the electrophoretic mobility of the peptides in which it occurs. The cytochrome from A. tumefaciens Apple 185 contains 125 amino acids of which 71 are identical in the protein from strain B2a. Together with cytochrome c-556 from the photosynthetic prokaryote Rhodopseudomonas palustris strain 2.1.37, the presently studied protein is the third known example of a monohaem class II cytochrome of the low-spin type having the single haem group covalently linked near the C terminus of the polypeptide chain. The only methionine residue in the Apple protein, methionine-13, is the most likely candidate to be the sixth haem ligand and therefore to be responsible for the low-spin character of the haem iron.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

Amino acid sequence of calmodulin from Euglena gracilis.

The complete amino acid sequence of calmodulin from Euglena gracilis was determined by isolation and sequence analyses of peptides derived from calmodulin by digestion with trypsin and Staphylococcus aureus V8 protease. Euglena calmodulin consists of 148 amino acid residues; it lacks tryptophan and cysteine and contains one tyrosine, three histidine and two NE-trimethyllysine residues/molecule of the protein. Its N-terminus was blocked with an acetyl group and C-terminal lysine was trimethylated. Euglena calmodulin is the first calmodulin so far examined in which the C-terminal lysine is trimethylated. The comparison of amino acid sequences between Euglena and human brain calmodulins indicated 17 amino acid substitutions in Euglena calmodulin.     

The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus.

The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.     

Thiobacillus ferrooxidans cytochrome c-552: purification and some of its molecular features

Soluble cytochrome c-552 was purified from Thiobacillus ferrooxidans to an electrophoretically homogeneous state. The cytochrome showed absorption peaks at 276, 411 and 523 nm in the oxidized form and peaks at 315, 417, 523 and 552 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 13,800 on the basis of the amino acid composition and heme content, and 14,000 from SDS-polyacrylamide gel electrophoresis analysis. Its midpoint redox potential at pH 7.0 was determined to be +0.36 V. The N-terminal amino acid sequence of the cytochrome was determined as follows: A-G-G-A-G-G-P-A-P-Y-R-I-S-?-D-?-M-V-?-S-G-M-P-G-. Ferrocytochrome c-552 was oxidized by the membrane fraction of T. ferrooxidans, and the oxidation rate was more rapid at pH 3.0 than at pH 6.5. Ferricytochrome c-552 was reduced by Fe(II)-cytochrome c oxidoreductase with Fe2+ at pH 3.5, while horse ferricytochrome c was not reduced by the enzyme under the same reaction conditions.