Structural studies of metalloenzyme complexes in acetogenic carbon fixation

Abstract

Acetogenic bacteria use the Wood-Ljungdahl carbon fixation pathway to produce cellular carbon from CO₂. This process requires several metalloenzymes that employ transition metals such as iron, nickel, and cobalt towards the production of acetyl-CoA, the final product. In one stage of the pathway, the cobalt-containing B₁₂ cofactor harbored by the corrinoid iron-sulfur protein (CFeSP) transfers a methyl group from methyltetrahydrofolate (CH₃-H₄folate), which is bound by a methyltransferase enzyme (MeTr), to a nickel-containing metallocluster called the A-cluster of the downstream enzyme, acetyl-CoA synthase (ACS). Such B12-dependent methyl transfer reactions require the construction of large, multimodular enzyme complexes whose threedimensional assemblies are, at present, largely uncharacterized. X-ray crystallography was used to solve the structure of a CFeSP/MeTr complex, the first crystal structure of a B12-dependent methyltransferase to depict all protein domains required for B12 binding, activation, protection, and catalysis. This structure, along with in crystallo activity data, illustrates how conformational movements, which can occur within protein crystals, enable the B12 cofactor to alternate between a sequestered conformation for cofactor protection and an active conformation for catalysis. Small-angle X-ray scattering (SAXS) experiments were also conducted to explore the quaternary composition of the complex in solution and revealed that multiple CFeSP/MeTr complexes can be formed. In another reaction of the Wood-Ljungdahl carbon fixation pathway, a nickel and iron containing metallocluster called the C-cluster of carbon monoxide dehydrogenase (CODH) reduces a second molecule of CO₂ to CO, an intermediate that is channeled to the ACS A-cluster. Although the structure of the C-cluster was first described a decade ago, its catalytic mechanism remained unresolved. To provide mechanistic insight into the chemistry employed at the C-cluster, crystal structures were determined with substrate and inhibitor molecules bound to the C-cluster of the CODH/ACS complex. These structures capture states of the C-cluster at key steps in the reaction and contribute to a consensus model for C-cluster chemistry. With structural descriptions for both CFeSP/MeTr and CODH/ACS complexes, this work has illuminated the molecular details for metalloenzyme complex assembly and catalysis in the acetogenic Wood-Ljungdahl carbon fixation pathway.