Structural insights into microbial one-carbon metabolic enzymes: Ni–Fe–S-dependent carbon monoxide dehydrogenases and acetyl-CoA synthases

Abstract

Carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS) enzymes play crucial rules in the global carbon cycle by catalyzing reversible carbon dioxide reduction and reversible acetyl-CoA synthesis, respectively. In some cases, CODHs are monofunctional, whereas in other cases CODHs form complexes with ACSs and their catalysis is coupled through an internal gas channel between the CODH and ACS active sites. These carbon-fixing enzymes are thought to be among the oldest on earth, dating back to the last universal common ancestor based on strong conservation of these enzymes between bacterial and archaeal domains of life. In this thesis, we present structural characterizations of bacterial and archaeal CODHs. Using xenon pressurization, we elucidate gas channel paths in a monofunctional CODH from bacteria through crystallographic studies. This structure provides the first experimental visualization of gas channels in a monofunctional CODH. We compare monofunctional CODH gas channels to the gas channels observed in bacterial CODH/ACS complexes and find monofunctional CODH gas channels are highly branched compared to in CODH/ACS complexes, wherein the specificity of the gas channel path is important for active site coupling. In methanogens, CODH and ACS catalysis are coupled, but a complex between these two enzymes was never previously visualized. The methanogenic CODH/ACS complex has been particularly mysterious because the methanogenic ACS lacks the domain that binds CODH in acetogens. In this work, we use cryogenic electron microscopy to capture the first-ever snapshot of an archaeal CODH/ACS complex. We observe a hydrophobic cavity between the CODH and ACS active sites that is rerouted relative to bacterial CODH/ACSs but conserved with a channel path in the monofunctional CODH. In another cryogenic electron microscopy structure of the archaeal CODH alone, we see that this hydrophobic cavity becomes plugged such that CO cannot leave CODH unless ACS is bound. This channel plugging mechanism is conserved with the channel plugging mechanism observed in the acetogenic CODH/ACS complex. This work advances our understanding of how CO is carried to and between active sites in CODH and ACS, and elucidates intriguing similarities between CODH/ACS complexes in acetogens and methanogens.