Use of Substrate Analogues and X-ray spectroscopy Reveals an all Ferrous C-Cluster in CO Dehydrogenase

Carbon monoxide (CO) dehydrogenase (CODH) plays a key role in prokaryotic one-carbon metabolism by detoxifying CO and by driving CO2 reduction coupled to ATP production in the Wood-Ljungdahl Pathway. Here we focus on a Ni-Fe CODH (CODH-II), with an active site C-cluster, which is a [NiFe4S4] cluster arranged as a [NiFe3S4] subcluster with an additional, unique pendant Fe, e.g., [Fe3S4-Feu]. It catalyzes the reversible reduction of CO2 to CO without the requirement for an overpotential and with insignificant proton reduction. The redox states associated with catalysis are defined as Cred1 and Cred2. Despite crystal structures with near 1.0 A resolution, it has been a long-standing question where the electrons in these catalytically relevant redox states are stored within the C cluster. Using X-ray absorption spectroscopy (XAS), EPR, and substrate-analogue binding measurements, we clarify the electronic structure of these catalytically active states in addition to the resting state of CODH. We rule out recent postulates that catalysis involves a Ni0 state, a metal-metal bond, or a hydride intermediate. We reveal that CODH rests in the diamagnetic Cox form, which contains Ni2+ and an oxidized [Fe3S4-Feu]2+ cluster. Then, the C-cluster undergoes reductive activation on Fe to form paramagnetic Cred1, which binds CO and analog cyanide. Generation of Cred2, which binds CO2 and its analog cyanate, involves two sequential valence-localized electron transfers, generating Ni1+ and then [Fe3S4-Feu], forming an all-ferrous cluster. Our work sheds light on how CODH avoids the thermodynamically unfavorable generation of a CO2 radical anion intermediate formed in other catalytic systems by stabilizing electron density in the heterometallic C-cluster. We also highlight the importance of high-resolution XAS and use of substrate analogs to reveal the sequential, valence-localized electron transfers that occur during redox-dependent CODH catalysis. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/700957v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1e8d3caorg.highwire.dtl.DTLVardef@a89cc9org.highwire.dtl.DTLVardef@1cd569dorg.highwire.dtl.DTLVardef@1f1668c_HPS_FORMAT_FIGEXP M_FIG C_FIG