Kinetic isotope effects of RuBisCO

The reactive enodiolate species is also sensitive to oxygen (O₂), which results in the dual carboxylase / oxygenase activity of RuBisCO.[13] This reaction is considered wasteful as it produces products (3-phosphoglycerate and 2-phosphoglycolate) that must be catabolized through photorespiration.[14] This process requires energy and is a missed-opportunity for CO₂ fixation, which results in the net loss of carbon fixation efficiency for the organism.[13] The dual carboxylase / oxygenase activity of RuBisCO is exacerbated by the fact that O₂ and CO₂ are small, relatively indistinguishable molecules that can bind only weakly, if at all, in Michaelis-Menten complexes.[15][16] There are four forms of RuBisCO (Form I, II, III, and IV), with Form I being the most abundantly used form. Form I is used extensively by higher plants, eukaryotic algae, cyanobacteria, and Pseudomonadota (formerly proteobacteria).[13] Form II is also used but much less widespread, and can be found in some species of Pseudomonadota and in dinoflagellates.[13] RuBisCOs from different photosynthetic organisms display varying abilities to distinguish between CO₂ and O₂. This property can be quantified and is termed "specificity" (S_c/o). A higher value of S_c/o means that a RuBisCO's carboxylase activity is greater than its oxygenase activity.

A generalized Michaelis-Menten curve.

The rate of carboxylation (V_C) is the rate that RuBisCO fixes CO₂ to RuBP under substrate saturated conditions.[14] A higher value of V_C corresponds to a higher rate of carboxylation. This rate of carboxylation can also be represented through its Michaelis-Menten constant K_C, with a higher value of K_C corresponding to a higher rate of carboxylation. V_C is represented by V_max, and K_C is represented as K_M in the generalized Michaelis-Menten curve. Although the rate of carboxylation varies among RuBisCO types, RuBisCO on average fixes only three molecules of CO₂ per second.[17] This is remarkably slow compared to typical enzyme catalytic rates, which usually catalyze reactions at the rate of thousands of molecules per second.[17]

Relationship between specificity and carboxylation rate of varying photosynthetic organisms.

It has been observed among natural RuBisCOs that an increased ability to distinguish between CO₂ and O₂ (larger values of S_c/o) corresponds with a decreased rate of carboxylation (lower values of V_C and K_C).[18] The variation and trade-off between S_c/o and K_C has been observed across all photosynthetic organisms, from photosynthetic bacteria and algae to higher plants.[18] Organisms using RuBisCOs with high values of V_C / K_C, and low values of S_c/o have localized RuBisCO to areas within the cell with artificially high local CO₂ concentrations. In cyanobacteria, concentrations of CO₂ are increased using a carboxysome, an icosahedral protein compartment about 100 nm in diameter that selectively uptakes bicarbonate and converts it to CO₂ in the presence of RuBisCO.[19] Organisms without a CCM, like certain plants, instead utilize RuBisCOs with high values of S_c/o and low values of V_C and K_C.[18] It has been theorized that groups with a CCM have been able to maximize K_C at the expense of decreasing S_c/o, because artificially enhancing the concentration of CO₂ would decrease the concentration of O₂ and remove the need for high CO₂ specificity. However, the opposite is true for organisms without a CCM, who must optimize S_c/o at the expense of K_C because O₂ is readily present in the atmosphere.

This trade-off between S_c/o and V_C or K_C observed in extant organisms suggest that RuBisCO has evolved through geologic time to be maximally optimized in its current, modern environment.[11][18] RuBisCO evolved over 2.5 billion years ago when atmospheric CO₂ concentrations were 300 to 600 times higher than present day concentrations, and oxygen concentrations were only 5-18% of present-day levels.[14] Therefore, because CO₂ was abundant and O₂ rare, there was no need for the ancestral RuBisCO enzyme to have high specificity. This is supported by the biochemical characterization of an ancestral RuBisCO enzyme, which has intermediate values of V_C and S_C/O between the extreme end-members.[14]

It has been theorized that this ecological trade-off is due to the form that 2-carboxy-3-keto-D-arabinitol 1,5-bisphophate in its transient transition state before cleaving into two 3PGA molecules.[11] The more closely the Mg²⁺-bound CO₂ moiety resembles the carboxylate group in 2-carboxy-3-keto-D-arabinitol 1,5-bisphophate, the greater the structural difference between the transition states of carboxylation and oxygenation.[11] The larger structural difference allows RuBisCO to better distinguish between CO₂ and O₂, resulting in larger values of S_c/o.[11] However, this increasing structural similarity between the transition state and the product state requires strong binding at the carboxyketone group, and this binding is so strong that the rate of cleavage into two product 3PGA molecules is slowed.[11] Therefore, an increased specificity for CO₂ over O₂ necessitates a lower overall rate of carboxylation. This theory implies that there is a physical chemistry limitation at the heart of Rubisco's active site, and may preclude any efforts to engineer a simultaneously more selective and faster Rubisco.[11][18]

Specificity of RuBisCO for CO₂ vs O₂ determines the extent of Carbon isotope fractionation.

S_c/o has been positively correlated with the magnitude of carbon isotope fractionation (represented by Δ¹³C), with larger values of S_c/o corresponding with a larger values of Δ¹³C.[11] It has been theorized that because increasing S_c/o means the transition state is more like the product, the O₂C---C-2 bond will be shorter, resulting in a higher overall potential energy & vibrational energy.[11] This creates a higher energy transition state, which makes it even harder for ¹³CO₂ (lower in the potential energy well than ¹²CO₂) to overcome the required activation energy.[11] The RuBisCOs used by varying photosynthetic organisms vary slightly in their enzyme structure, and this enzyme structure results in varying transition states. This diversity in enzyme structure is reflected in the resulting Δ¹³C values measured from different photosynthetic organisms. However, overlap exists between the Δ¹³C values of different groups because the carbon isotope values measured are generally of the entire organism, and not just its RuBisCO enzyme. Many other factors, including growth rate and the isotopic composition of the starting substrate, can affect the carbon isotope values of whole organism and cause the spread seen in C isotope measurements.[20][21]