# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 2 Materials and Methods
### Assay Preparation and Execution  
Prior to running the KIE assay, the activity of bovine erythrocytes CA (Sigma Aldrich; St. Louis, MO, USA C3934) was checked following manufacturer guidelines [39]. We found a value of 3368 W-A units/mg protein, which exceeded the product stated value of \(\geq\)2000 W-A units/mg protein, and so we proceeded to use this active CA enzyme prep in the KIE assay.  
Glass sampling vials with septum tops ('Exetainer,' 12 mL, Labco, Lampeter, UK) were prepared. First, three external standards were prepared by weighing out Carrara marble standards (CIT_CM2013, \(\delta^{13}\)C = 2.0 \(\pm\) 0.1%) into individual exetaers. Standards were then sealed within each tube, purged with He gas for 5 min, and then acidified by needle injection with concentrated phosphoric acid (42% _v/v_) (Sigma-Aldrich, St. Louis, MO, USA). Then, three HCO\({}_{3}\)\({}^{-}\) substrate exetaers were also sealed, purged with He gas, acidified by needle injection of phosphoric acid to convert HCO\({}_{3}\)\({}^{-}\) to CO\({}_{2}\), and placed in a 70 \({}^{\circ}\)C water bath for at least 20 min. Finally, 22 exetainer sampling vials were prepared for the rubiscos (12 for L\({}_{8}\), 10 for L\({}_{8}\)S\({}_{8}\)). All sampling tubes were first sealed and purged with He gas for 5 min, and then injected with ~1 mL of anhydrous phosphoric acid (Sigma-Aldrich, St. Louis, MO, USA). The phosphoric acid both stops the reaction progress and converts all dissolved inorganic carbon species into CO\({}_{2}\) for analysis.  
Next, the reaction assay for each rubisco was prepared. First, a CA stock solution was made by dissolving bovine erythrocytes CA into DI water. Next, an RuBP stock solution was made by dissolving D-Ribulose 1,5-bisphosphate sodium salt hydrate (Sigma Aldrich; St. Louis, MO, USA R0878) in DI water. Then, one drop of concentrated hydrochloric acid (38% _v/v_) was added to 20 mL of autoclaved DI water while it was simultaneously stirredwith a stir bar and vigorously bubbled with N\({}_{2}\) gas for 10 min to remove any residual HCO\({}_{3}\)- or CO\({}_{2}\). Then, while N\({}_{2}\) gas was blown over the surface of the solution to inhibit O\({}_{2}\), reagents were added to create a final concentration of 100 mM bicine, 30 mM MgCl\({}_{2}\), 1 mM dithiothreitol (DTT) (St. Louis, MO, USA), and 6.25 mM NaHCO\({}_{3}\) (St. Louis, MO, USA). pH was adjusted to 8.5 with NaOH and HCl. CA from the CA stock was added, and then either the L\({}_{8}\) or L\({}_{8}\)S\({}_{8}\) rubisco was added to the solution. We used 0.996 mg of L\({}_{8}\)S\({}_{8}\) and 1.18 mg of L\({}_{8}\) rubisco. The solution was gently bubbled with N\({}_{2}\) gas for 10 min while rubisco 'activated.' While the solution was bubbling, the syringes used for each rubisco assay were rinsed with ethanol and water. We used a separate 25 mL gas-tight syringe with a sample-locking needle for each rubisco (Ref #66326, Model 1025 SL SYR, Hamilton Company, Reno, NV, USA).  
RuBP was then added to each reaction assay and mixed through pipetting and swirling. This entire solution was then quickly transferred to a gas-tight syringe. The first time point (t = 0 min) was taken as quickly as possible after transfer. To sample, \(\sim\)1 mL of the reaction assay was injected into the pre-prepared sampling exetaer containing phosphoric acid. Each assay was sampled 10-12 times over 390 min.  
A control was run in a separate experiment, where all the assay components were mixed together with the exception of a rubisco enzyme. The \(\delta^{13}\)C of the measured headspace did not change appreciably during this time period, with \(\delta^{13}\)C = \(-\)0.42 \(\pm\) 0.03% at 0 min and \(\delta^{13}\)C = \(-\)0.55 \(\pm\) 0.03% at 277 min. The absolute values of these measurements reflect the \(\delta^{13}\)C of the substrate used on that experimental day and cannot be related to the data shown here.