Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on early Earth. As the early atmosphere became oxygenated, these microorganisms needed protection from oxygen, which was especially important for those organisms that relied on enzymes with oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation, 4-hydroxybutyryl-CoA dehydratase (4HBD), adapted from anoxic to oxic conditions. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical 4Fe-4S cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments support our findings that restricting access to the active site is key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition.