• BMB Reports
• /
• 제29권4호
• /
• pp.300-307
• /
• 1996

ATP and ADP are potential regulators of mitochondtial respiration and at physiological concentrations they affect the rate of electron transfer between cytochrome c and cytochrome c oxidase. The electron transfer, however, depends on the electrostatic interaction between the two proteins. In order to exclude any nonspecific ionic effects by these polyvalent nucleotides, we used 2'-O-(2,4,6)trinitro(TNP)-derivatives of ATP and ADP which have three orders of magnitude higher affinity for cytochrome c oxidase. A simple titration of the fluorescence intensity of TNP by cytochrome c oxidase showed a binding stoichiometry of 2:1 cytochrome c:cytochrome c oxidase. Higher ionic strength was required for TNP-ATP than for TNP-ADP to be dissociated from cytochrome c oxidase, indicating that the negative charges on the phosphate group are at least partially responsible for the binding. In both spectrophotometric and polarographic assays, addition of ATP (and ADP to a less extent) showed an enhanced cytochrome c oxidase activity. Both electron paramagnetic resonance and fluorescence spectra indicate that there is no Significant change in the cytochrome c-cytochrome c oxidase interaction. Instead, reduction levels of the cytochromes at steadystate suggest that the increased activity of nucleotide-bound cytochrome c oxidase is due to faster electron transfer from cytochrome ${\alpha}$ to cytochrome ${\alpha}_3$, which is known to be the fate limiting step in the oxygen reduction by cytochrome c oxidase.

• BMB Reports
• /
• 제30권6호
• /
• pp.397-402
• /
• 1997

A thiol-specific spin label was attached to cysteine-102 of yeast cytochrome c and electron paramagnetic resonance (EPR) spectra were measured as a function of added cytochrome c oxidase concentration. The intensity decreased due to line broadening as cytochrome c formed a complex with cytochrome c oxidase and reached a minimum when the ratio of cytochrome c to cytochrome c oxidase became one. Replacement of either Lys-72 or Lys-87 of cytochrome c by Glu did not result in a significant change in binding affinity. Interestingly the K72E mutant, unlike K87E, had a much lower rate of electron transfer than the wild type. These results indicate that many positively charged residues as a group participate in complex formation but Lys-72 might be important for cytochrome c to be locked in an orientation for an efficient electron transfer. A stoichiometry of 1 was also confirmed by optical absorption of the cytochrome c-cytochrome c oxidase complex which had been run through a gel chromatography cloumn to remove unbound cytochrome c. The EPR spectrum of this 1:1 complex, however, was a mixture of two components. This explains a biphasic kinetics for a single binding site on cytochrome c oxidase without invoking conformational transition.

• BMB Reports
• /
• 제39권4호
• /
• pp.452-456
• /
• 2006

To elaborate the peroxidase activity of cytochrome c in the generation of free radicals from $H_2O_2$, the mechanism of DNA cleavage mediated by the cytochrome c/$H_2O_2$ system was investigated. When plasmid DNA was incubated with cytochrome c and $H_2O_2$, the cleavage of DNA was proportional to the cytochrome c and $H_2O_2$ concentrations. Radical scavengers, such as azide, mannitol, and ethanol, significantly inhibited the cytochrome c/$H_2O_2$ system-mediated DNA cleavage. These results indicated that free radicals might participate in the DNA cleavage by the cytochrome c and $H_2O_2$ system. Incubation of cytochrome c with $H_2O_2$ resulted in a time-dependent release of iron ions from the cytochrome c molecule. During the incubation of deoxyribose with cytochrome c and $H_2O_2$, the damage to deoxyribose increased in a time-dependent manner, suggesting that the released iron ions may participate in a Fenton-like reaction to produce $\cdot$OH radicals that may cause the DNA cleavage. Evidence that the iron-specific chelator, desferoxamine (DFX), prevented the DNA cleavage induced by the cytochrome c/$H_2O_2$ system supports this mechanism. Thus we suggest that DNA cleavage is mediated via the generation of $\cdot$OH by a combination of the peroxidase reaction of cytochrome c and the Fenton-like reaction of free iron ions released from oxidatively damaged cytochrome c in the cytochrome c/$H_2O_2$ system.

• 미생물학회지
• /
• 제30권2호
• /
• pp.101-107
• /
• 1992

화학 영양성으로 배양한 Rps. gelatinosa 에서 2회의 시토크롬 c 친화성 크로마토그래피와 DEAE-Sephacel 이온 교환 크로마토그래피 등 3 단계의 크로마토그래피를 수행하여 시토로콤 c 산화효소를 정제하였다. 정제된 시토크롬 c 산화효소는 Sephacryl S-300 에 의한 분자걍이 약 110,000 Da 이고 SDS-gel 전기영동에 의한 분자량이 약 52.000 Da 으로써 이량체일 것으로 보인다. 전제된 시토크롬 c 산화효소는 온도데 매우 불안정하고 말 심장 시토크롬 c 를 기질로 사용했을때 Km 값은 $20\mu$M, Vmax 값은 44unit/mg prot. 이며 pH 6.4 의 효소방응 최적 pH 와 25.deg.C 의 최적 온도를 보였다. 환원된 시토크롬 c 산화효소는 554, 523, 421 nm 에서 .alpha., .betha. soret 흡수대를 보였고 chromatophore 에서와 마찬가지로 KCN 과 $NaN_{3}$ 에 의해서는 효소 활성도가 저해를 받았지만 CO 와 antimycin A, myxothiazol 에 의해서는 효소 활성도가 저해를 받지 않았다. 빛을 에너지원으로 배양하거나 또는 화학영양성으로 배양하든지 모두 시토크롬 c-551 이 생성되었고 환원된 시토크롬 c-551 은 시토크롬 c 산화효소에 의해 산화되었다. 시토크롬 c-551 을 기질고 이용하였을 때 시토크롬 c 산화효소의 Km 값은 $26\mu$M 이었고 Vmax 값은 31.unit./mg prot. 로써 말심장의 시토크롬 c 를 기질로 이용할때 보다 오히려 낮았다. 이와 같은 결과로 보아 화학 영양성은 배양한 Rhodopseudomonas gelatinosa 에서 호흡에 의한 전자전달은 시토크롬 c-551 이 시토크롬 $bc_{1}$ 복합체로 부터 전자를 받아 b-형 시토크롬 c 산화효소에 전자를 전달해 주고 최정적으로 산소를 환원시킬 것으로 생각된다.

• 미생물학회지
• /
• 제29권4호
• /
• pp.243-249
• /
• 1991

The chromatophore from the chemotrophically grown facultative anaerobic photosynthetic bacterium, Rhodopseudomonas gelatinosa ATCC 17013 was isolated through stepwise sucrose gradient centrifugation. The isolated chromatophore showed high activities of the cytochrome $bc_{1}$ complex and cytochrome c oxidase. The activity of cytochrome $bc_{1}$ complex was completely inhibited by .5$\mu$M antimycin A,10$\mu$M myxothiazol, and that of cytochrome c oxidase was completely inhibited by .$50\mu$M KCM and $100\mu$M $NaN_{3}$but not inhibited by carbon monoxie. The activity of cytochrome c oxidase of th chromatophore was increased by addition of ionophores or protonophores. The reduced-oxidised difference sspectrum of cytochrome $bc_{1}$ complex isolated by affivity chromatography showed the absorption maxima at 553 nm(shoulder at 547 nm), 520 nm, and 418.5 nm, on the other hand, that of cytochrome c oxidase showed .alpha., .betha. and soret peaks at 554 nm, 523 nm, and 421 nm, respectively. The cytochrome c oxidase from chemotrophically grown Rhodopseudomonas gelatinosa seems to be a b-type cytochrome c oxidase.

• Bulletin of the Korean Chemical Society
• /
• 제28권1호
• /
• pp.77-80
• /
• 2007

As neurofilament proteins are major cytoskeletal components of neuron, abnormality of neurofilament is proposed in brain with neurodegenerative disorders such as Parkinson's disease (PD). Since oxidative stress might play a critical role in altering normal brain proteins, we investigated the oxidative modification of neurofilament-L (NF-L) induced by the reaction of cytochrome c with H2O2. When NF-L was incubated with cytochrome c and H2O2, the protein aggregation was increased in cytochrome c and H2O2 concentrationsdependent manner. Radical scavengers, azide, formate and N-acetyl cysteine, prevented the aggregation of NFL induced by the cytochrome c/H2O2 system. The formations of carbonyl group and dityrosine were obtained in cytochrome c/H2O2-mediated NF-L aggregates. Iron specific chelator, desferoxamine, prevented the cytochrome c/H2O2 system-mediated NF-L aggregation. These results suggest that the cytochrome c/H2O2 system may be related to abnormal aggregation of NF-L which may be involved in the pathogenesis of PD and related disorders.

• Bulletin of the Korean Chemical Society
• /
• 제27권6호
• /
• pp.830-834
• /
• 2006

Lipid peroxidation induced by the reaction of cytochrome c with $H_2O_2$ was investigated. When linoleic acid micelles or phosphatidyl choline liposomes were incubated with cytochrome c and $H_2O_2$, lipid peroxidation was increased in cytochrome c and $H_2O_2$ concentrations-dependent manner. Radical scavengers, azide, formate and ethanol prevented lipid peroxidation induced by the cytochrome c/$H_2O_2$ system. Iron specific chelator, desferoxamine also prevented the cytochrome c/$H_2O_2$ system-mediated lipid peroxidation. These results suggest that lipid peroxidation may be induced by the cytochrome c/$H_2O_2$ system via the generation of free radicals. Carnosine, homocarnosine and anserine are present in the muscle and brain of many animals and human. Previous studies show that these compounds have an antioxidant function. In the present study, carnosine, homocarnosine and anserine significantly prevented the cytochrome c/$H_2O_2$ system-mediated lipid peroxidation. Carnosine and related compounds also inhibited the free radical-generating activity of cytochrome c. The results suggest that carnosine, homocarnosine and anserine may prevent lipid peroxidation induced by the cytochrome c/$H_2O_2$ system through a free radical scavenging.

• Applied Microscopy
• /
• 제38권2호
• /
• pp.125-133
• /
• 2008

Mitochondria 내막의 cytochrome-c-oxidase는 세포의 에너지 생합성에 중요한 요소이며, 세포자멸사와 각종세포의 병리학적 현상과 밀접한 연관성이 있는 전자전달계효소로 알려져 있다. Porin 단백은 mitochondria 내막과 외막에 분포하는 효소단백으로 전자전달계효소 형성과 ATP 운반에 관여하는 것으로 알려져 있다. 따라서 면역현미경법을 사용하여 cytochrome-c-oxidase의 분포와 porin 단백과의 연관성을 확인하여 mitochondria의 cristae에 분포하는 cytochrome-c-oxidase의 형성과 변화를 알아보고자 하였다. Cardiac muscle tissue의 sarcoplasm에는 많은 수의 mitochondria가 분포하며, cytochrome-c-oxidase가 풍부한 mitochondria와 porin 단백이 풍부한 mitochondria로 구별되었다. Cytochrome-c-oxidase가 풍부한 mitochondria는 porin 단백이 빈약하고 porin 단백이 풍부한 mitochondria는 cytochrome-c-oxidase가 소량 포함되어 있는 것으로 관찰되었다. 심근조직의 부위에 따라 근형질에 분포하는 mitochondria에 cytochrome-c-oxidase가 풍부한 mitochondria와 porin 단백이 풍부한 mitochondria가 각각 상이하게 분포하였다. 이상의 결과로 미성숙 mitochondria는 많은 양의 porin 단백을 함유하여 근형질로부터 단백질 소단위를 mitochondria 막내로 운반하여 cytochrome-c-oxidase를 형성시키고 mitochondria가 성숙하면서 ATP를 운반할 최소한 양의 porin 단백만을 남기고 소멸되는 것으로 추측된다.

• 한국식품과학회지
• /
• 제19권1호
• /
• pp.50-53
• /
• 1987

Cytochrome C가 돼지지방의 산화에 미치는 영향을 조사하고자 돼지등 지방, cytochrome C, 아스콜빈산 및 아질산염을 사용하여 pH 5.8에서 지방산화도를 TBA방법으로 측정하였다. Cytochrome c첨가랑이 증가할수록 지방산화는 증가하였고, 산화증가정도는 Cytochrome c 첨가량이 증가할수록 감소하였다. 아스콜빈산이나 아질산염은 Cytochrome c의 지방산화촉진효과를 억제하지못하였으나, 두가지를 동시에 첨가하였을때는 지방산화촉진을 억제하였다. 결과적으로 가공육제품에 Cytoch-rome 가 다량함유된 염통을 첨가하여도 육제품 품질에 나쁜 영향을 주지 않을 것으로 사료된다.

• BMB Reports
• /
• 제31권4호
• /
• pp.307-327
• /
• 1998

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme which catalyzes the reduction of hydrogen peroxide to water using two equivalents of ferrocytochrome c. The CcP/cytochrome c system has many features which make it a very useful model for detailed investigation of heme protein structure/function relationships including activation of hydrogen peroxide, protein-protein interactions, and long-range electron transfer. Both CcP and cytochrome c are single heme, single subunit proteins of modest size. High-resolution crystallographic structures of both proteins, of one-to-one complexes of the two proteins, and a number of active-site mutants are available. Site-directed mutagenesis studies indicate that the distal histidine in CcP is primarily responsible for rapid utilization of hydrogen peroxide implying significantly different properties of the distal histidine in the peroxidases compared to the globins. CcP and cytochrome c bind to form a dynamic one-to-one complex. The binding is largely electrostatic in nature with a small, unfavorable enthalpy of binding and a large positive entropy change upon complex formation. The cytochrome c-binding site on CcP has been mapped in solution by measuring the binding affinities between cytochrome c and a number of CcP surface mutations. The binding site for cytochrome c in solution is consistent with the crystallographic structure of the one-to-one complex. Evidence for the involvement of a second, low-affinity cytochrome c-binding site on CcP in long-range electron transfer between the two proteins is reviewed.