# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 4 Discussion
### Presence or Absence of RbcS External to Active Site May Influence KIE
Unexpectedly, the L8 rubisco fractionates roughly 5% less than that of the L8S8 rubisco (16.25 \(\pm\) 1.36% vs. 22.42 \(\pm\) 2.37%, respectively). The specificity of the L8 rubisco is indeed less than that of the L8S8 (36.1 \(\pm\) 0.9 vs. 56.1 \(\pm\) 1.3, respectively, [30]) but this may be a coincidence because that prediction is based on a theory reliant on rubisco's active site which the small subunit does not directly impact. Our comparative study suggests the tantalizing hypothesis that the small subunit increases rubisco KIEs. However, Form I' has only recently been discovered [30] and only a limited number of sequences exist. Future work consisting of dual CO2 and O2 KIE measurements of other novel Form I' rubiscos compared to Form I rubiscos, across a range of assay parameters, will be needed for a more robust comparative study. Potentially, comparative studies of extant L8 vs. L8S8 rubiscos could be complemented with experiments using ancestral rubiscos demonstrated to not require RbcS-RbcL interactions [51] that would allow one to effectively strip the small subunit from an L8S8 rubisco and measure its effect on the KIE. Similarly, pairings of one RbcL sequence with various RbcS sequences of tobacco rubiscos [67] would allow one to test how various small subunits affect the KIE in Form I (L8S8) rubiscos. Alternately, it has been shown that mutations distal from the active site affecting oligomerization can affect enzyme kinetics, which is somewhat analogous to losing RbcS in that does not directly interact with the active site. KIE measurements from such rubiscos may also help shed help shed light on the relationship between RbcS, specificity, and KIE [68]. Therefore, it remains an open question what structural and biochemical aspects of rubisco may also affect KIEs in addition to active site and transition state theory mechanisms. Supports Prior Work Positing That Rubisco KIEs Vary across Phylogeny in the Modern Day and across Time  
Our work supports previous work showing that the rubisco KIE varies across phylogeny in the modern day, though with the caveats that few unique rubiscos have been measured, there is variation across experiments, and the vast majority of measurements are from Form I rubiscos (Figure 1B, and see [13,37] for recent compilation across phylogeny). A smaller KIE measured from one novel Form I' rubisco, in comparison to the bacterial Form I rubiscos, supports this observation, though more measurements across the Form I clade are needed to quantify any potential in-clade variation. In addition, if we view L8 as an evolutionary'missing link' between the evolution of L2 and L8S8 rubiscos, this measurement supports the idea that rubisco KIE may have varied across evolutionary time. Prior work has explored this question by measuring the KIE of a putative Precambrian ancestral Form IB rubisco reconstructed using a combination of phylogenetic and molecular biology techniques [54]; that study found the ancestral rubisco to fractionate less than its modern counterpart (17.23 \(\pm\) 0.61% vs. 25.18 \(\pm\) 0.31%, respectively) [50]. Interestingly, the Form I' and putative ancestral Form IB rubisco have similar, lower KIE values (16.25 \(\pm\) 1.36% vs. 17.23 \(\pm\) 0.61%, respectively) compared to most modern Form I rubiscos (roughly 20-30%; for recent review see [13]). This supports prior predictions that the KIE should have varied over geologic time in response to changing pCO2, though that prediction was based on an assumption of inverse correlation between specificity (selected for by changing CO2/O2 levels) and KIE [15]. This implies that the KIE of ancestral rubiscos may have been lower than modern rubiscos today, though this is a tentative hypothesis that, by necessity, relies on ancestral enzyme reconstruction and comparative biology techniques instead of direct measurements of 'true' ancestral enzymes.