Step In Cytochrome Research Articles

A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as FeH1II(NO2-), where FeH1 represents the ccNiR active site. FeH1II(NO2-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {FeH1NO}7; ccNiR was not reduced beyond the {FeH1NO}7 state. The midpoint potentials for sequential reduction of FeH1III(NO2-) to FeH1II(NO2-) and then to {FeH1NO}7 were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. FeH1II(NO2-) does not accumulate at equilibrium because its reduction to {FeH1NO}7 is so much easier than the reduction of FeH1III(NO2-) to FeH1II(NO2-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {FeH1NO}7 is exceedingly slow (k ∼ 0.001 s-1), but it is somewhat faster (k ∼ 0.050 s-1) while FeH1III(NO2-) is being reduced to {FeH1NO}7; then, the release of NO• from the undetectable transient {FeH1NO}6 can compete with reduction of {FeH1NO}6 to {FeH1NO}7. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {FeH1NO}7 over the more labile {FeH1NO}6.

Hyperstability and crystal structure of cytochromec555from hyperthermophilicAquifex aeolicus

In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c(555) (AA c(555)) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c(555) exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c(555), which was determined using the multiple anomalous dispersion technique at 1.15 A resolution, included a unique 14-residue extra helix, while the side-chain interactions of several amino-acid residues responsible for the stability of HT c(552) were conserved in AA c(555). The side chain of the Met61 residue in the extra helix was aligned towards the haem, forming a coordination bond between the Met S and haem Fe atoms. In other cytochromes c the corresponding regions always form Omega loops which also include the haem-liganding Met residue and are known to be involved in the initial step in cytochrome c denaturation. The formation of the extra helix in AA c(555) results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c(555) and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment.

ABC transporter‐mediated release of a haem chaperone allows cytochrome c biogenesis

Although organisms from all kingdoms have either the system I or II cytochrome c biogenesis pathway, it has remained a mystery as to why these two distinct pathways have developed. We have previously shown evidence that the system I pathway has a higher affinity for haem than system II for cytochrome c biogenesis. Here, we show the mechanism by which the system I pathway can utilize haem at low levels. The mechanism involves an ATP-binding cassette (ABC) transporter that is required for release of the periplasmic haem chaperone CcmE to the last step of cytochrome c assembly. This ABC transporter is composed of the ABC subunit CcmA, and two membrane proteins, CcmB and CcmC. In the absence of CcmA or CcmB, holo(haem)CcmE binds to CcmC in a stable dead-end complex, indicating high affinity binding of haem to CcmC. Expression of CcmA and CcmB facilitates formation of the CcmA2B1C1 complex and ATP-dependent release of holoCcmE. We propose that the CcmA2B1C1 complex represents a new subgroup within the ABC transporter superfamily that functions to release a chaperone.

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase

Cytochrome c oxidase (Cyt cO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn 2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides Cyt cO with O 2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn 2+. The results show that addition of Zn 2+ at concentrations of ≤250 μM to the outside of the vesicles did not alter the transition rates between intermediates P R ( P 3 ) → F 3 → O 4 . However, proton pumping was impaired specifically during the P 3 → F 3 , but not during the F 3 → O 4 transition at Zn 2+ concentrations of ≤ 25 μM. Furthermore, proton pumping during the P 3 → F 3 transition was typically impaired with the “as isolated” Cyt cO, which was found to contain Zn 2+ ions at μM concentration. As has already been shown, Zn 2+ was also found to obstruct proton uptake during the P 3 → F 3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a K I of ∼ 1 μM for this binding site. In conclusion, the results show that Zn 2+ ions bind on both sides of Cyt cO and that binding of Zn 2+ at the proton output side selectively impairs proton release during the P 3 → F 3 transition.

シトクロムcの折れ畳み初期に形成する疎水性クラスターの性質

シトクロムcの折れ畳み初期に形成する疎水性クラスターの性質

Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen

Cytochrome c oxidase is a membrane-bound enzyme that catalyses the reduction of O 2 to H 2O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn 2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo-ferryl (P r→F) and the oxo-ferryl to oxidised (F→O) transitions. The Zn 2+ binding resulted in a decrease of the F→O rate from 820 s −1 (no Zn 2+) to a saturating value of ∼360 s −1 with an apparent K D of ∼2.6 μM. The P r→F rate (∼10 4 s −1 before addition of Zn 2+) decreased more slowly with increasing Zn 2+ concentration and a K D of ∼120 μM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the P r→F and F→O transitions are associated with proton uptake through the D-pathway, a Zn 2+-binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co-ordinate Zn 2+.

The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase

The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1 − mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa 3 and of a cytochrome c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex.

Interaction of NADPH-adrenoferredoxin reductase with NADP+ and adrenoferredoxin. Equilibrium and dynamic properties investigated by proton nuclear magnetic resonance.

NADPH-adrenoferredoxin reductase, a flavoprotein from bovine adrenocortical mitochondria, has been investigated to elucidate the equilibrium and dynamic properties of the interaction with NADP+ and adrenoferredoxin (adrenodoxin) using proton NMR spectroscopy. The line width of the signals from NADP+ depends on the presence of the reductase. The off rate constant of NADP+ from the reductase is estimated to be about 15-20 s-1 on the basis of line width measurements. No appreciable difference in off rate is detected between adenine and nicotinamide moieties of NADP+. Transferred nuclear Overhauser effect experiments for NADP+ indicate the time-dependent magnetization transfer profiles with a long lag phase. The proton NMR spectra during the titration of the reductase with adrenodoxin reveal that the reductase possesses distinct binding sites for both NADP+ and adrenodoxin. The sharp resonances in the aromatic region due to His-10 and His-62 of adrenodoxin were utilized as a probe to explore the interaction with the reductase. IN the mixture of adrenodoxin and the reductase at the mol ratio of 6:1, T1 values of the histidine residue in adrenodoxin were measured by the inversion recovery method. At low ionic strength, T1 values of the resonances are not affected in the presence or absence of the reductase. In the presence of the reductase, T1 values of resonances resulting from the histidine residues become shorter as the concentration of KCl increases because of rapid exchange between bound and free states. At low ionic strength (10 mM phosphate buffer), the off rate from the reductase is estimated to be less than about 4 s-1. The off rate of adrenodoxin from the reductase could be the rate-limiting step in cytochrome c reductase activity at low ionic strength.

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.

Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy.

The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.

ChemInform Abstract: Timing of the Radical Recombination Step in Cytochrome P‐450 Catalysis with Ring‐Strained Probes.

AbstractNortricyclane, methylcyclopropane, and bicyclo(2.1.0)pentane are used to probe the catalytic mechanism of microsomal cytochrome P‐450.

Rate-limiting steps in cytochrome P-450-catalysed reactions: studies on isotope effects in the O-de-ethylation of 7-ethoxycoumarin.

Primary deuterium isotope effects ranging from 2 to 6 were measured for the O-de-ethylation of 7-ethoxycoumarin catalysed by microsomal and purified cytochrome P-450 isozymes. Interpretation of the observed deuterium isotope effect in terms of the contribution of the C-H bond-cleavage step to the overall rate requires the determination of the intrinsic isotope effect. Evidence is presented that at least one irreversible step occurs before C-H bond cleavage which kinetically divides the cytochrome P-450 reaction cycle into two portions. Intermolecular primary isotope effects are shown to provide kinetic information only about steps following the irreversible step. Consequently, the isotope effect may be a measure of the rate limitation imposed by the C-H bond-cleavage step in the sequence of events following the irreversible step but be totally independent of other slow steps preceding the irreversible step. These results indicate that multiple rate-limiting steps may exist in cytochrome P-450-catalysed reactions.