# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 1 Introduction  
Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) is a keystone enzyme linking Earth's inorganic and organic carbon cycles, which makes it a prime target for bioengineering associated with food systems and carbon sequestration. It is the most abundant protein on Earth today [1] because it catalyzes the essential carbon fixation step in one of the most ecologically dominant carbon-fixing metabolisms, the Calvin Benson Bassham (CBB) cycle in oxygenic photosynthesis [2]. Rubisco and oxygenic photosynthesis form the basis of our food web in terrestrial and marine systems because both eukaryotic and bacterial primary producers utilize rubisco to convert inorganic carbon (CO\({}_{2}\) and HCO\({}_{3}\)\({}^{-}\)) into biomass that is then consumed by heterotrophs up the food chain. In addition, the annual flux of CO\({}_{2}\) fixed by rubisco is very large, representing the single most massive flux in the global carbon cycle. Gross primary productivity (GPP), which accounts for all forms of carbon fixation but is vastly dominated by oxygenic photosynthesis, is \(\approx\)120 Gt C yr\({}^{-1}\) in terrestrial [3] and \(\approx\)100 Gt C yr\({}^{-1}\) in marine environments [1, 4], compared to \(\approx\)10 Gt C yr\({}^{-1}\) emitted of anthropogenic fossil CO\({}_{2}\)[5]. Therefore, multiple efforts exist to engineer a 'better' rubisco that fixes more CO\({}_{2}\) in order to increase crop yields and sequester anthropogenic CO\({}_{2}\), among many other motivations (see [6] for review). However, these bioengineering approaches are informed to a degree by our current understanding of rubisco's evolutionary history, which itself is based on our understandingof past Earth environments. These evolutionary questions largely center on the canonical paradox that, despite being a central metabolism enzyme, rubisco is: (i)'slow,' and (ii) 'confused' because it can fix O\({}_{2}\) instead of CO\({}_{2}\)[7], which invokes a salvage pathway that costs ATP, reducing power, and carbon [8]. This paradox is usually resolved by considering the atmospheric composition when rubisco first evolved more than 2.5 billion years ago, when CO\({}_{2}\) was much higher (potentially up to \(\approx\)20x present atmospheric levels in the Pre-cambrian [9]) and O\({}_{2}\) only existed at trace levels [2]. However, in a Shakespearean tragedy, once rubisco was incorporated into the greater metabolism of oxygenic photosynthesis, it poisoned the very world it came from--successful CO\({}_{2}\) fixation was coupled with oxygenation that permanently changed the atmosphere to one where O\({}_{2}\) is dominant (\(\approx\)20%) and CO\({}_{2}\) is trace (\(\approx\)0.04%). Now saddled with a rubisco evolved from a chemical world that no longer exists, diverse land plants, algae, and Cyanobacteria have independently evolved complex CO\({}_{2}\) concentrating mechanisms (CCMs) that effectively hyper-concentrate CO\({}_{2}\) at the expense of O\({}_{2}\) around rubisco [10]--in effect, replicating the ancient atmosphere within their own cells. Those without CCMs (e.g., C3 plants) instead accommodate the low carboxylation rate by producing this enzyme at such high concentrations that up to 65% of all soluble protein in leaf extracts is just rubisco [11]. This narrative, contingent on our understanding of the geologic carbon cycle, suggests either that rubisco is an 'accident' of evolutionary history, or that it is truly the optimal enzyme designed by evolution for a difficult task. Therefore, a better understanding of the evolutionary history of this enzyme is useful for rubisco engineering efforts. Rubisco is also notable because it displays a large carbon kinetic isotope effect (KIE) where it preferentially fixes \({}^{12}\)CO\({}_{2}\) over \({}^{13}\)CO\({}_{2}\) due to the rate of carboxylation being slightly faster for \({}^{12}\)CO\({}_{2}\)[12].