# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 3 Results  
_L\({}_{g}\) Rubisco Has Smaller KIE than Its L\({}_{g}\)S\({}_{8}\) Counterpart_  
The KIE of the L\({}_{8}\) rubisco is \(\approx\)5% less than that of the L\({}_{8}\)S\({}_{8}\) rubisco (16.25 \(\pm\) 1.36% vs. 22.42 \(\pm\) 2.37%, respectively; Table 1). We note that there is variation among KIE measurements of similar or the same strains. Prior measurements which we compare our \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Strain** & **Rubisco** & **KIE (\%)** & **V\({}_{\mathrm{C}}\) (s\({}^{-1}\))** & **K\({}_{\mathrm{C}}\) (\(\upmu\)M)** & **S\({}_{\mathrm{CO}}\)** & **V\({}_{\mathrm{O}}\) (s\({}^{-1}\))** & **K\({}_{\mathrm{O}}\) (\(\upmu\)M)** \\ \hline _Synechococcus elongatus_ & L\({}_{8}\)S\({}_{8}\) & 22.42 \(\pm\) 2.37 & 14.3 \(\pm\) 0.71 & 235 \(\pm\) 20.0 & 56.1 \(\pm\) 1.3 & 1.10 & 983 \(\pm\) 81 \\ PCC6301 & & & & & & & \\ \hline _Candidatus_ Promineofilum & L\({}_{8}\) & 16.25 \(\pm\) 1.36 & 2.23 \(\pm\) 0.04 & 22.2 \(\pm\) 9.7 & 36.1 \(\pm\) 0.9 & 1.11 & 401 \(\pm\) 115 \\ breve & & & & & & \\ \hline \end{tabular}
\end{table} 
Table 1: KIE and non-isotopic kinetic measurements from L\({}_{8}\) vs. L\({}_{8}\)S\({}_{8}\) rubisco. data against (Figure 1, Table S2) are bacterial (Form II, Form I') or Cyanbacterial (Form I) rubisco measurements, where a pure enzyme, substrate-depletion assay such as ours was performed on well-characterized strains where rubisco was obtained through expression and subsequent purification from _E. coli_. We also included one Form II measurement from a _Riftia pachyptila_ symbiont, _Candidatus_ Endoriftia Persephone [44], where rubisco was purified from the host trophosome because at the time of the measurement the symbiont could not be cultured separately from the host, though a complete genome has recently been published [45]. Therefore, we did not include measurements where a non-native bacterial rubisco was expressed by another organism in vivo and KIE calculated by extrapolating ratios of intracellular to extracellular CO\({}_{2}\)[46], nor measurements from plants or the _Solemya velum_ symbiont because it is not a member of the Cyanobacteria [36]. It has been proposed and measured that rubisco KLEs vary with pH, temperature, and metal ion concentrations [47; 48], yet other studies contradict this claim [49] and have instead proposed that much of the variation in the literature reflects experimental uncertainties rather than intrinsic variations in KIE [16]. This study and [50] measured an L\({}_{8}\)S\({}_{8}\) rubisco KIE from _Synechococcus elongatus_ PCC6301 and 7942, respectively (identical RbcL and RbcS sequences) in similar assay conditions but found values that are similar but do not overlap in uncertainty, supporting the conclusion that variations in reported KIE values are due to experimental uncertainty rather than intrinsic enzymatic variations. However, the KIEs presented in Figure 1 were measured in assays that span a range of pH, temperature, and MgCl\({}_{2}\) concentrations (Table S2), notably with increasing MgCl\({}_{2}\) concentration corresponding with increasing KIEs measured in the Form II rubisco by [25]. Because of the lack of repeated, rigorous measurements of multiple rubisco KLEs across variations relevant parameters (i.e., pH, temperature, metalation), it is difficult to conclude what is causing the variation in KIE values across studies. Therefore, we can only conclude that the L\({}_{8}\) rubisco KIE is less (by roughly 5%) than its L\({}_{8}\)S\({}_{8}\) counterpart measured in this study, and less than the range of L\({}_{8}\)S\({}_{8}\) rubiscos measured from previous studies. Figure 1: Form I’ rubisco fractionated less than both Form II and Form I rubiscos, and cannot be explained by prior model relating specificity and KIE. (**A**) KIE (%) for relevant bacterial Form II (L\({}_{2}\)), Form I’ (L\({}_{8}\)), and Cyanobacterial Form IA/B (L\({}_{8}\)S\({}_{8}\)) rubiscos with representative rubisco structures below; Protein Data Bank (PDB) codes from left to right: 5 RUB, 6 URA, 1 RBL.