• 분석과학
• /
• 제8권4호
• /
• pp.869-876
• /
• 1995

The active sites of the nickel and iron-containing enzyme, carbon monoxide dehydrogenase (CODH) from clostridium thermoaceticum were investigated using Electron Paramagnetic Resonance (EPR) technique. CODH exhibits several spectral features called NiFeC, $g_{ave}=1.82$, $g_{ave}=1.86$. FCII signals which are originated from different clusters in this enzyme. CODH is know to catalyze two different kinds of reactions - acetyl-CoA synthesis and CO oxidation. The acetyl-CoA synthesis activity can be followed by monitoring CO/acetyl-CoA exchange. The addition of 1,10-phenanthroline (phen) to CODH selectively destroyed the CO/acetyl-CoA exchange activity and eliminated the NiFeC signal completely. CO oxidation activity and other EPR signals were unaffected. Such behavior demonstrates that CODH has two distinct active sites and that the NiFe complex is only responsible for the CO/acctyl-CoA exchange activity. Phen caused the removal of only 30% of Ni in the NiFe complex ($0.3Ni/{\alpha}{\beta}$) as shown by the quantitative metal analysis. The phen-treated CODH could be reactivated fully by incubation In $Ni^{2+}$ solution. Radioactive $^{63}Ni^{2+}$ was used to quantitate the amount of the $Ni^{2+}$ incorporated into phen-treated enzyme and showed that the amount was the same as the removed by the phen treatment. i.e. $0.3Ni/{\alpha}{\beta}$. This indicates that only 30% of NiFe complexes are labile and responsible for the CO/acctyl-CoA exchange activity, the other 70% are non-labile and have no exchange activity. This is the first clear evidence that the NiFe complex is heterogencous and labile and non-labile Ni sites arc interacting differently with substrates and chelating agents like phen.

• Journal of Microbiology and Biotechnology
• /
• 제9권4호
• /
• pp.404-413
• /
• 1999

Methanopyrus kandleri is a hyperthermophilic methanogen that represents one of the most heat-resistant organisms: the maximum growth temperature of M. kandleri is $110^{\circ}C$. A random sequence analysis of the genomic DNA of M. kandleri has been performed to obtain genomic information. More than 200 unique sequence tags were obtained and compared with the sequences in the GenBank and PIR databases. About 30% of the analyzed tags showed strong sequence similarity to previously identified genes involved in various cellular processes such as biosynthesis, transport, methanogenesis, or metabolism. When statistics relating to the frequency of codons were examined, the sequenced open reading frames showed highly biased codon usage and a high content of charged amino acids. Among the identified genes, a homologue of the catalytic subunit of carbon monoxide dehydrogenase (CODH) that reduces $CO_2$ to CO was cloned and sequenced in order to examine its detailed gene structure. The cloned gene includes consensus promoters. The amino acid sequence of the cloned gene shows a strong homology with the CODH genes from methanogenic Archaea, especially in the presumed binding sites for Fe-S centers.

• 전기화학회지
• /
• 제11권3호
• /
• pp.165-169
• /
• 2008

CODH(Carbon Monoxide Dehydrogenase)에 의한 이산화탄소 환원에 있어서 작업전극을 유리탄소전극을 사용한 경우와 금전극을 사용한 경우를 비교하여 그 영향을 관찰하였다. 금전극을 사용한 경우에는 수소발생과 섞이기 때문에 전기분해의 전위를 잘 선택해야 효율적인 이산화탄소의 환원 반응을 관찰할 수 있는데 반하여, 유리탄소전극은 금전극보다 수소 환원에 대한 과전압이 크기 때문에 -650 mV vs. NHE 까지도 중성수용액에서 수소발생 없이, 효율적인 이산화탄소의 환원을 관찰할 수 있었다. CODH를 이용한 이산화탄소의 환원에는 가해주는 전기분해 전위가 큰 영향을 미침을 알 수 있었는데, $-570{\sim}600\;mV$ vs. NHE 근처가 가장 효율적임을 알 수 있었고 이보다 더 음의 전위를 걸어주었을 때는 효소활성의 감소 및 수소발생이 복합적으로 영향을 미쳐 일산화탄소 생성의 전류효율이 급격히 감소함을 알 수 있었다.

• 전기화학회지
• /
• 제10권4호
• /
• pp.265-269
• /
• 2007

일산화탄소탈수소화효소를 이용한 이산화탄소의 전기화학적 환원 반응의 최적화를 위해 pH와 온도의 영향을 살펴보았다. pH는 이산화탄소의 환원반응 용이성과 효소의 안정도 효과가 함께 반영되는 6.3에서 최적의 조건을 보였고, 온도는 이산화탄소의 용해도가 적어지더라도 효소의 활성이 가장 좋은 60도에서 최적 조건을 보였다. 일산화탄소탈수소화효소의 안정성 및 활성이 가장 중요한 조건임을 알 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제13권5호
• /
• pp.717-724
• /
• 2003

A newly isolated Citrobacter sp. Y19 catalyzes the CO-dependent $H_2$ production (biological water-gas shift reaction) by the actions of CO dehydrogenase (CODH) and hydrogenase. Y 19 requires $O_2$ for fast growth, but its $H_2$ production activity is significantly inhibited by $O_2$. In the present study, the effect of $O_2$ on the activities of CODH ard hydrogenase was investigated quantitatively in both whole cells and broken cells, based on CO-dependent or methyl viologen (MV)-dependent $H_2$ production in addition to CO-dependent MV reduction. In crude cell extracts, CODH activity was mostly found in the soluble fraction. Inactivation of CODH and hydrogenase activities by $O_2$ followed the first-order decay kinetics, and the dependence of the rate constants on $O_2$ partial pressure could be expressed by the Michaelis-Menten equation. In whole cells, the maximum deactivation rate constants ($k_{d,max}$ of hydrogenase and CODH were quite similar: $0.07{\pm}0.03 min^{-1}\;and\;0.10{\pm}0.04 min^{-1}$, respectively. However, the first-order rate constant ($k_{d,max}/K_s$) of CODH ($0.25\;min^{-1}\;atm^{-1}$) at low $O_2$ partial pressures was about 3-fold higher than that of the hydrogenase, since the half-saturation constant ($K_s$) of CODH was about half of that of hydrogenase. In broken cells, both enzymes became significantly more sensitive to $O_2$ compared to the unbroken cells, while $k_{d,max}/K_s$ increased 37-fold for hydrogenase and 6.7-fold for CODH. When whole cells were incubated under anaerobic conditions after being exposed to air for 1 h, hydrogenase activity was recovered more than 90% in 2 h suggesting that the deactivation of hydrogenase by $O_2$ was reversible. On the contrary, CODH activity was not recovered once deactivated by $O_2$ and the only way to recover the activity was to synthesize new CODH. This study indicates that $O_2$ sensitivity of $H_2$ production activity of Citrobacter sp. Y19 is an important drawback as in other $H_2-producing$ bactria.