EPR characterization of a high‐spin system in carbon monoxide dehydrogenase from Methanothrix soehngenii

Abstract

Carbon monoxide dehydrogenase and methyl‐coenzyme M reductase were purified from ⁶¹Nienriched and natural‐abundance nickel‐grown cells of the methanogenic archae Methanothrix soehngenii. The nickel‐EPR signal from cofactor F‐430 is metyl‐CoM reductase was of substoichiometric intensity and exhibited near‐axial symmetry with g= 2.153, 2.221 and resolved porphinoid nitrogen superhyperfine splittings of ∼ 1 mT. In the spectrum from ⁶¹Ni‐enriched enzyme a wellresolved parallel I= 3/2 nickel hyperfine splitting was observed, A_‖= 4.4 mT. From a computer simulation of this spectrum the final enrichment in ⁶¹Ni was estimated to be 69%, while the original enrichment of the nickel metal was 87%. Carbon monoxide dehydrogenase isolated from the same batch exhibited four different EPR spectra. However, in none of these signals could any splitting or broadening from ⁶¹Ni be detected. Also, the characteristic g= 2.08 EPR signals found in some other carbon monoxide dehydrogenases and ascribed to a Ni‐Fe‐C complex, was never observed by us under any conditions of detection (4 to 100 K) and incubation in the presence of ferricyanide, dithionite, CO, coenzyme A, or acetyll‐coenzyme A. Novel, high‐spin EPR weas found in the oxidized enzyme with effective g‐values at g= 14.5, 9.6, 5.5, 4.6, 4.2, 3.8. The lines at g= 14.5 and 5.5 were tentatively ascribed to an S= 9/2 system (∼ 0.3 spins/αβ) with rhombicity E/D= 0.047 and D < 0. The other signals were assigned to an S= 5/2 system (0.1 spins/αβ) with E/D= 0.27. Both sets of signals disappear upon reduction with E_m.7.5=–280 mV. With a very similar reduction potential, E_m.7.5=–261 mV, an S= ‐261 mV, an S= 1/2 signal (0.1 spins/αβ) appears with the unusual g‐tensor 2.005, 1.894, 1.733. Upon further lowering of the potential the putative double cubane signal also appears. At a potential E∼ ‐320 mV the double cubane is only reduced by a few percent and this allows the detection of individual cubane EPR not subjected to dipolar interaction; a single spectral component is observed with g‐tensor 2.048, 1.943, 1.894.