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-Fe_u]. 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 C_red1 and C_red2. 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 C_ox form, which contains Ni²⁺ and an oxidized [Fe₃S₄-Fe_u]²⁺ cluster. Then, the C-cluster undergoes reductive activation on Fe to form paramagnetic Cred1, which binds CO and analog cyanide. Generation of C_red2, which binds CO₂ and its analog cyanate, involves two sequential valence-localized electron transfers, generating Ni¹⁺ and then [Fe₃S₄-Fe_u], 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.