Discovery of a labile nickel ion required for CO/acetyl-CoA exchange activity in the NiFe complex of carbon monoxide dehydrogenase

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1992

OSTI ID:7112925

Shin, W S

The oxygen-sensitive, nickel and iron-containing enzyme carbon monoxide dehydrogenase (CODH) from clostridium thermoaceticum was purified without any significant loss of activity. The careful examinations of activities, metal contents, and EPR properties of CODH during its purification showed that the low spin intensities of CODH EPR signals were not coming from purification procedures, oxygen damage, or metal ion dissociation. Oxygen-damaged enzyme could be separated from active enzyme chromatographically. Exposure to oxygen during the purification lowered the overall yield but not the specific activity of the finally purified enzyme. CODH does not require zinc or a third type of protein subunit for CO oxidation or CO/acetyl-CoA exchange activities. CODH has a low Hase activity. CO binding properties of the NiFe complex were examined by the titration of CO with dithionite-reduced CODH. One molecule of CO is needed to develop full NiFeC signal intensity. CO binds tightly to the NiFe complex. This suggests that the low spin intensity of the NiFeC signal is not caused by substoichiometric binding of CO. The addition of phen to CODH selectively destroyed the CO/acetyl-CoA exchange activity and eliminated the NiFeC signal completely. Such behavior demonstrates that CODH has two distinct active sites and that the NiFe complex is only responsible for the CO/acetyl-CoA exchange activity. Phen caused the removal of only 30% of Ni in the NiFe complex. Only 30% of NiFe complexes are labile and responsible for the CO/acetyl-CoA exchange activity. This is the first clear evidence that the NiFe complex is heterogeneous. The NiFe complex contains as many as 5--9 irons. Fe[sup 2+] could be incorporated into the vacant Ni site of NiFe complex. The affinity of Fe[sup 2+] was much less than Ni[sup 2+] and resulting EPR signal resembled the NiFeC signal. Fe[sup 2+]-incorporated CODH has negligible CO/acetyl-CoA exchange activity.

• Research Organization: Texas A and M Univ., College Station, TX (United States)
• OSTI ID: 7112925
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400201 -- Chemical & Physicochemical Properties
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMISTRY
CARBON COMPOUNDS
CARBON MONOXIDE
CARBON OXIDES
CATALYSIS
CATALYTIC EFFECTS
CHALCOGENIDES
CHARGED PARTICLES
CHEMICAL REACTIONS
CHEMISTRY
CLOSTRIDIUM
COENZYMES
COMPLEXES
DEHYDROGENATION
ELECTRON SPIN RESONANCE
ENZYME ACTIVITY
ENZYMES
HETEROGENEOUS CATALYSIS
IONS
IRON COMPLEXES
MAGNETIC RESONANCE
MICROORGANISMS
NICKEL COMPLEXES
NICKEL IONS
ORGANIC COMPOUNDS
OXIDES
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PROTEINS
PURIFICATION
RESONANCE
TRANSITION ELEMENT COMPLEXES