A relationship between prokaryote and eukaryote observed in Nitrobacter agilis cytochromes AA3 and C

Cytochrome aa3 (cytochrome c oxidase) and cytochrome c were purified from Nitrobacter agilis, and some of their properties were compared with those of the respective counterparts of eukaryote from the evolutionary point of view. N. agilis cytochrome aa3 has many functional and structural properties similar to those of eukaryotic cytochrome aa3, although its molecule is composed of only two kinds of subunits unlike the eukaryotic cytochrome which is composed of 7 kinds of subunits. N. agilis cytochrome c is homologous to eukaryotic cytochrome c; 50 amino acid residues of the bacterial cytochrome c are identical with those of horse cytochrome c. It reacts with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochrome c does. So far as based on the molecular features of cytochromes aa3 and c, N. agilis appears to be one of the organisms which may link in evolution prokaryote to eukaryote.     

A comparison between Nitrosomas europaea and Thiobacillus novellus on the basis of their oxidation systems of inorganic compounds.

Nitrosomonas europaea and Thiobacillus novellus were compared with each other on the basis of the biochemical properties of their inorganic compound-oxidizing systems. Cytochromes c of the two organisms differ considerably from each other; N. europaea cytochrome c-552 belongs to the "bacterial-type" cytochrome c, while T. nouellus cytochrome c-550 resembles eucaryolic cytochrome c. The specificity of cytochrome oxidase for cytochrome c as the electron donor is different between the two organisms; T novellus oxidase reacts rapidly with cytochromes c of the organisms which seem to be higher than the organisms whose cytochromes c react rapidly with N. europaea oxidase. On the basis of these facts, N. europaea seems to be older organism than T. novellus in terms of evolution.     

Catalytic properties of cytochrome c oxidase purified from Nitrosomonas europaea

Cytochrome c oxidase of Nitrosomonas europaea reacts with not only the native cytochrome c (N. europaea cytochrome c-552) but also horse and yeast cytochromes c. The effects on its reactivity of various reagents were very different between the reactions with the native and eukaryotic cytochromes c as the electron donors. The oxidation of eukaryotic ferrocytochrome c by the oxidase was activated by addition of anionic detergents such as sodium dodecyl sulfate and sodium cholate, and anionic phospholipids such as cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, while the reaction was not activated by Triton X-100, Tween 20, or phosphatidylcholine. However, the reaction with the native cytochrome c of the enzyme was hardly affected by any of the detergents and phospholipids mentioned above, while it was activated by the presence of poly-L-lysine.     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.     

High-resolution three-dimensional structure of horse heart cytochrome c

The 1.94 A resolution three-dimensional structure of oxidized horse heart cytochrome c has been elucidated and refined to a final R-factor of 0.17. This has allowed for a detailed assessment of the structural features of this protein, including the presence of secondary structure, hydrogen-bonding patterns and heme geometry. A comprehensive analysis of the structural differences between horse heart cytochrome c and those other eukaryotic cytochromes c for which high-resolution structures are available (yeast iso-1, tuna, rice) has also been completed. Significant conformational differences between these proteins occur in three regions and primarily involve residues 22 to 27, 41 to 43 and 56 to 57. The first of these variable regions is part of a surface beta-loop, whilst the latter two are located together adjacent to the heme group. This study also demonstrates that, in horse cytochrome c, the side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a conformation comparable to that found in other cytochromes c. The positioning of this residue does not therefore appear to be oxidation-state-dependent. In total, five water molecules occupy conserved positions in the structures of horse heart, yeast iso-1, tuna and rice cytochromes c. Three of these are on the surface of the protein, serving to stabilize local polypeptide chain conformations. The remaining two are internally located. One of these mediates a charged interaction between the invariant residue Arg38 and a nearby heme propionate. The other is more centrally buried near the heme iron atom and is hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78. It is shown that this latter water molecule shifts in a consistent manner upon change in oxidation state if cytochrome c structures from various sources are compared. The conservation of this structural feature and its close proximity to the heme iron atom strongly implicate this internal water molecule as having a functional role in the mechanism of action of cytochrome c.     

Cytochromec and evolution of the energy acquiring system

The reactivity of cytochromesc derived from various organisms withPseudomonas aeruginosa nitrite reductase and cow cytochrome oxidase has been studied.Generally, cytochromesc isolated from primitive organisms react very rapidly with the bacterial nitrite reductase but do not react with cow cytochrome oxidase while those from higher organisms react poorly with the nitrite reductase but react very rapidly with the animal oxidase. The reactivity of cytochromec with the bacterial nitrite reductase reflects very well the evolutionary position of the organism from which it is isolated, while that with cow cytochrome oxidase seems to be related to the extent of adaptation of the parent organism to molecular oxygen. The results obtained in the present investigation suggests that cytochromec molecule which reacts very rapidly with the bacterial nitrite reductase but does not react with cow cytochrome oxidase has evolved to that which reacts very poorly with the nitrite reductuase but reacts very rapidly with the animal oxidase. It is also inferred that the evolution of cytochromec molecule may be caused by the evolution of cytochrome oxidase, and that the latter may be intimately related to genesis of molecular oxygen in the biosphere.Special Symposium on Photochemistry and the Origins of Life, Sixth International Congress on Photobiology, Bochum, Germany.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a1c1 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 a1c1 and aa3-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 cytochrome c could be pushed through prompt removal by cytochrome aa3 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 a1c1 with NO2- in vivo.     

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.     

Effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c on reactions with oxidase, reductase and peroxidase

The effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c (Kuo, L.M. and Davies, H.C. (1983) Mol. Immunol. 20, 827-838) in the reactions of the cytochromes c with cytochrome c oxidase, reductase and peroxidase were studied. Spectrophotometric assays were employed, under conditions where binding of cytochrome c to the enzymes appears to be rate-limiting. Less than stoichiometric amounts of antibodies to P. denitrificans cytochrome c added to the cytochrome rendered some of it nonoxidizable or nonreducible by the P. denitrificans membrane-bound electron transport system and decreased the rate constant with the remaining cytochrome c. The antibodies appear to affect both electron transport reactions (blocking effects) with the oxidase and reductase and binding effects (effects on rate constants) and to distinguish between the two. Different ratios of antibody site to cytochrome c gave different extents of blocking of the reductase as compared with the oxidase reaction. Differences were also apparent in the effect of these antibodies on the reaction of yeast peroxidase and the oxidase with the P. denitrificans cytochrome c. Antibodies to bovine and P. denitrificans cytochromes c had considerably less effect on the reactions of the bovine cytochrome with bovine oxidase and reductase. One antibody was inhibitory to the oxidase reaction with bovine cytochrome c, but not to that with the reductase. Also, an antibody which inhibited the oxidase reaction had no effect on the reaction with yeast peroxidase. The data give evidence that the interaction areas on cytochrome c for oxidase and reductase and peroxidase are not identical, although they may be nearby.     

Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus

Cytochrome oxidase from T. thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R. M., Choc, M. G., Tarr, G. E., Findling, K. L., and Fee, J. A. (1983) J. Biol. Chem. 258, 112-123). We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T. thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD). These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions. Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme. The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3. When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized. The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme.     

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.     

Characterization of recombinant horse cytochrome c synthesized with the assistance of Escherichia coli cytochrome c maturation factors

Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains. The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism. It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E. coli cytochrome c maturation factors achieves a native-like structure and stability. The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c. Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result.     

Comparison of the structures of various eukaryotic ferricytochromes c and ferrocytochromes and their antigenic differences

The nuclear magnetic resonance spectra of various eukaryotic ferricytochromes c and ferrocytochromes c are described. The proteins from the species donkey, cow, dog, rabbit, chicken and pigeon were investigated. The conformations of these proteins detected by NMR were compared to those of horse and tuna cytochromes c and in some cases small differences were found. These differences in structure were shown to correlate with antigenic differences between the various proteins.     

Comparative kinetic studies of cytochromes c in reactions with mitochondrial cytochrome c oxidase and reductase.

Kinetic studies of the reactions of selected eukaryotic and prokaryotic cytochromes c with mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase (EC 1.9.3.1) using a standardized complex IV preparation from beef heart are reported. Data on reactions with NADH-linked cytochrome c reductase (complexes I and III) are included. The concentration ranges employed provide a basis for quantitative demonstration of a general rate law applicable to oxidase reactions of cytochrome c of greatly differing reactivities. Results are interpreted on the basis of a modified Minnaert mechanism (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23), assuming productive complex formation between cytochrome c and free oxidase in addition to further complex binding of a second cytochrome c molecule to the initially formed oxidase complex. Kinetic constants so obtained are consistent with the assumption that binding is the dominant parameter in reactivity, and can be rationalized most simply on this basis.     

Proteins of the thermophilic fungus Humicola lanuginosa. II. Some physicochemical properties of a cytochrome c

A cytochrome c from Humicola lanuginosa is unique among eukaryotic cytochromes c in having phenylalanine as Residue 74. This protein has certain properties which differ from those of other cytochromes c to which it is generally similar. The Humicola cytochrome c is as stable as horse heart cytochrome c in urea, but more stable than both horse heart and yeast cytochromes c in acidic and alkaline conditions. Spectrophotometric titration of the four tyrosyl residues of the Humicola protein was nonsigmoidal with a pK_app of 11.4. Solvent perturbation difference spectra indicate that 50% of the tyrosyl residues are exposed to solvent in the native protein, and that the single tryptophanyl and all four tyrosyl residues become exposed in 8 m urea. Certain unusual features in both the optical rotatory dispersion and circular dichroism spectra in the 290-250-nm region are tentatively attributed to the substitution of phenylalanine for tyrosine at position 74.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

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.     

COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.