# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 1 Introduction
This effect is typically reported in delta (\(\delta^{13}\)C) and epsilon (\(\varepsilon\)) notation in units of per mille (%), where \(\delta^{13}\)C = [\({}^{13}\)R\({}_{\rm{sat}}\)/\({}^{13}\)R\({}_{\rm{ref}}\) - 1]*1000 and \({}^{13}\)R is the ratio of \({}^{13}\)C/\({}^{12}\)C in the sample or reference, respectively. \(\varepsilon\) is roughly the difference in \(\delta^{13}\)C between the product and the reactant (\(\varepsilon_{\rm{Rubisco}}\approx\delta^{13}\)C\({}_{\rm{3 PG}}-\delta^{13}\)C\({}_{\rm{CO2}}\)). Thirteen unique rubisco KIES (\(\varepsilon_{\rm{Rubisco}}\) values) have been measured across a limited range of phylogenies and species, but measurements so far indicate that rubisco fractionates at roughly 20-30% (for a recent review see [13]). This KIE is useful because it allows one to track mass flux through complex systems in both modern and ancient environments [14], and because it may give insight into non-isotopic enzyme kinetics [15]. Since all biomass is ultimately synthesized from 3 PG in autotrophs utilizing the CBB cycle, rubisco's KIE is inherited by bulk biomass such that organic carbon is also relatively depleted in \({}^{13}\)C relative to inorganic carbon. Therefore, when incorporated into larger metabolic models of carbon fixation, rubisco KIES have facilitated the estimation of water use efficiency in plants [16], the efficiency of carbon fixation in bacterial and eukaryotic algae [17], the contribution of terrestrial plants to global GPP [18], and the proportion of C3 vs. C4 plants in mammalian diets [19], among many other examples. Similarly, in ancient environments, it has been used to estimate paleo atmospheric CO\({}_{2}\) levels [20; 21], track the inorganic and organic carbon cycle through time [22], and the diet of ancient mammals [23]. In addition, rubisco KIES have been used to support interpretation of important non-isotopic kinetic parameters such as the inverse correlation between specificity for CO\({}_{2}\) over O\({}_{2}\) (S\({}_{\rm{C/O}}\)) and rate of carboxylation (V\({}_{\rm{C}}\)) [15]. Therefore, knowing the KIES of many rubiscos is valuable because it facilitates empirical measurements of mass flux in many systems, natural and engineered, where other measurements may be difficult. However, the landscape of rubisco evolution and its effect on KIE has not been well characterized. This is particularly true in Cyanobacteria, the organism within which rubisco and oxygenic photosynthesis is thought to have evolved. Most rubisco KIES have been measured for Form IB rubiscos from plants, and in Cyanobacteria, only one Form IA and one Form IB rubisco KIE have been measured ([24; 25], for a recent review see [13]). This is particularly important for reconstructing paleo pCO\({}_{2}\) levels because direct measurements of the atmosphere from ice core records only extend back \(\approx\)1 million years [26], so for the remainder of Earth's 4.567 billion year history we must rely on indirect measurements such as the carbon isotope record: globally assembled measurements of \(\delta^{13}\)C in the inorganic or organic carbon bearing phases of sedimentary rocks [27]. Interpretation of these records relies on geochemical models, largely based on extant modern organisms, that incorporate the rubisco KIE to explain most of the offset in \(\delta^{13}\)C between inorganic and organic carbon pools (see [28] for recent review of current models). These models inform our understanding of ancient atmospheres which in turn can influence our ideas of rubisco evolution in the past and engineering strategies in the present. It is therefore critical that we better understand the evolution of rubisco's KIE through time because it underlies many assumptions we make when interpreting both the past and present. We therefore tried to address this gap in knowledge by studying one key example, a Form I rubisco that lacks the small subunit.