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 61Ni-enriched and natural-abundance nickel-grown cells of the methanogenic archae Methanothrix soehngenii. The nickel-EPR signal from cofactor F-430 in methyl-CoM reductase was of substoichiometric intensity and exhibited near-axial symmetry with g = 2.153, 2.221 and resolved porphinoid nitrogen superhyperfine splittings of z 1 mT. In the spectrum from 61Ni-enriched enzyme a wellresolved parallel I = 3/2 nickel hyperfine splitting was observed, All = 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 "Ni be detected. Also, the characteristic g = 2.08 EPR signal 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 acetyl-coenzyme A. Novel, high-spin EPR was 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 = 912 system (~0 . 3 spinslap) with rhombicity EID = 0.047 and D < 0.The other signals were assigned to an S = 512 system (0.1 spinslag) with E / D = 0.27. Both sets of signals disappear upon reduction with Em, 7 . 5 = -280 mV. With a very similar reduction potential, Em,,,5 = -261 mV, an S = l j 2 signal (0.1 spinslap) 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 z -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.Carbon monoxide dehydrogenase (CDH) is the central enzyme in the Wood pathway for autotrophic acetylcoenzyme A (CoASAc) synthesis by homoacetogenic bacteria [I -31. In these bacteria the enzyme catalyzes several reactions, the most important being the reduction of C 0 2 to CO and the condensation of a methyl group bound to CDH with CO and CoASH to CoASAc [4 -61. CDH also plays an important role in acetate degradation by methanogenic archae, where the enzyme catalyzes the oxidation of CO to COz and the cleavage of CoASAc to a methyl, carbonyl and CoASH moiety [7-101. Although C D H activity has opposite functions in the metabolisms of these two microbial groups, on the molecular level the enzymes have several properties in common. CDH constitutes up to 2 -5% of the soluble protein and this reflects its metabolic importance. The enzyme has a tetrameric structure ( M , Y )~, but it can be part of a complex [4, lo]. The genes for the subunits are clustered in a opcronlike struct...

Electrochemical study of the redox properties of [2Fe‐2S] ferredoxins Evidence for superreduction of the Rieske [2Fe‐2S] cluster

“…This cluster appears to contain some six iron atoms [24]. Spectroscopic, sequence data, and X-ray structural evidence points to the possibility of similar 'superclusters' in other ironsulfur redox enzymes, viz sulfite reductase [25], CO dehydrogenase [26,27], and nitrogenase [28,29]. This information combined with what is known on the redox properties of small, electron transfer iron-sulfur proteins [4] has led us to hypothesize the following seniority scheme for the redox properties of biological iron-sulfur clusters [30][31][32].…”

Multi‐frequency EPR and high‐resolution Mössbauer spectroscopy of a putative [6Fe‐6S] prismane‐cluster‐containing protein from Desulfovibrio vulgaris (Hildenborough)

“…The unusually high system spin S = 912 has thus far been tentatively identified in three iron-sulfur enzymes: dis-similatory sulfite reductase [6], carbon-monoxide dehydrogenase [8] and the FeMo-protein of nitrogenase [5]. Several synthetic iron cluster compounds with S = 9/2 ground states have also recently been described [34 -361.…”

Purification and biochemical characterization of a putative [6Fe‐6S] prismane‐cluster‐containing protein from Desulfovibrio vulgaris (Hildenborough)

“…This hypothesis explains the absence of classical ironsulfur clusters and the presence of unusual EPR spectra in multielectron redox proteins with a high Fe/S content by proposing the existence of superclusters of more than four magnetically coupled Fe ions, giving rise to unprecedented superspin ( S 2 712) paramagnetism. This concept particularly addresses the high Fe/S content of complex enzymes such as hydrogenase [l 11, nitrogenase component 1 [12], dissimilatory sulfite reductase [13] and carbonmonoxide dehydrogenase [14]. The existence of larger iron-sulfur clusters, e.g.…”