Reactivity of a Paramagnetic Enzyme−CO Adduct in Acetyl-CoA Synthesis and Cleavage

• Published in: Biochemistry (Easton) Vol. 35; no. 2; pp. 593 - 600
• Main Authors: Grahame, David A, Khangulov, Sergei, DeMoll, Edward
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16-01-1996
• Subjects: Acetyl Coenzyme A - biosynthesis; Acetyl Coenzyme A - chemistry; Acetyl Coenzyme A - metabolism; Aldehyde Oxidoreductases - chemistry; Aldehyde Oxidoreductases - metabolism; Carbon Monoxide - chemistry; Electron Spin Resonance Spectroscopy; Methanosarcina barkeri; Methanosarcina barkeri - enzymology; Molecular Structure; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Spectrophotometry; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9511494

Partial reactions of acetyl-CoA cleavage by the Methanosarcina barkeri acetyl-CoA decarbonylase synthase enzyme complex were investigated by UV−visible and electron paramagnetic resonance (EPR) spectroscopy. Reaction of the enzyme complex with carbon monoxide generated an EPR-detectable adduct with principal g values of 2.089, 2.076, and 2.028, and line widths of 13.76, 16.65, and 5.41 G, respectively. The EPR signal intensity was dependent upon both enzyme and carbon monoxide concentration. A second signal with g av = 2.050 was generated by storage of the CO-exposed enzyme for 17 months at −70 °C. Reaction of the enzyme complex with low levels of CO caused reduction of the enzyme complex, but did not result in immediate formation of the NiFeC signal (designated NiFeC based on isotopic substitution studies carried out by others in analogous systems from Clostridium thermoaceticum and Methanosarcina thermophila). Further addition of CO generated the NiFeC signal, and the signal amplitude then increased progressively with increasing CO concentration. UV−visible spectra showed that enzyme Fe-S and corrinoid centers were already fully reduced at levels of CO significantly lower than needed for maximal EPR signal intensity. This result indicated that the EPR signal is formed by reaction of the reduced enzyme with CO (or a reduced one-carbon species), rather than with a one-carbon unit at the oxidation level of CO2. Addition of coenzyme A, acetyl-CoA, or tetrahydrosarcinapterin had no effect on the EPR signal. In contrast, addition of N 5-methyltetrahydrosarcinapterin (CH3-H4SPt) abolished the EPR signal. EPR spectra recorded at 20−21 K revealed that reaction with CH3-H4SPt affects only the enzyme NiFeC signal, and does not influence other EPR-detectable Fe-S center(s). The results suggest that the enzyme−CO adduct reacts with CH3-H4SPt to form an EPR-silent enzyme−acetyl species. Preincubation of the enzyme complex with CO and CH3-H4SPt, both of which were required, produced an approximately 44-fold increase in the turnover rate of acetyl-CoA synthesis. The relevance of these findings to mechanisms involving possible reductive methylation of the enzyme and/or acetyl-enzyme formation is discussed.