Seasonal variation in foliar carbon exchange in Pinus radiata and Populus deltoides: respiration acclimates fully to changes in temperature but photosynthesis does not

We investigated seasonal variation in dark respiration and photosynthesis by measuring gas exchange characteristics on Pinus radiata and Populus deltoides under field conditions each month for 1 year. The field site in the South Island of New Zealand is characterized by large day-to-day and seasonal changes in air temperature. The rate of foliar respiration at a base temperature of 10 °C ( R ₁₀) in both pine and poplar was found to be greater during autumn and winter and displayed a strong downward adjustment in warmer months. The sensitivity of instantaneous leaf respiration to a 10 °C increase in temperature ( Q ₁₀) was also greater during the winter period. The net effect of this strong acclimation was that the long-term temperature response of respiration was essentially flat over a wide range of ambient temperatures. Seasonal changes in photosynthesis were sensitive to temperature but largely independent of leaf nitrogen concentration or stomatal conductance. Over the range of day time growth temperatures (5–32 °C), we did not observe strong evidence of photosynthetic acclimation to temperature, and the long-term responses of photosynthetic parameters to ambient temperature were similar to previously published instantaneous responses. The ratio of foliar respiration to photosynthetic capacity ( R _d/ A _sat) was significantly greater in winter than in spring/summer. This indicates that there is little likelihood that respiration would be stimulated significantly in either of these species with moderate increases in temperature – in fact net carbon uptake was favoured at moderately higher temperatures. Model calculations demonstrate that failing to account for strong thermal acclimation of leaf respiration influences determinations of leaf carbon exchange significantly, especially for the evergreen conifer.     

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of α-keto acid-dependent dioxygenases

The α-ketoglutate (α-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe^II, α-KG and dioxygen for catalysis, with the α-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the α-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe^IVO intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O₂ to generate this species is the decarboxylation of the α-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the α-keto acid to Fe^II and the presence of a 5C Fe^II site for the O₂ reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate α-KG coordination and a 5C Fe^II site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an 10⁵-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n → π^* transition of the HPPD/Fe^II/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the α-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO₂ is disfavored.     

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of alpha-keto acid-dependent dioxygenases

The alpha-ketoglutate (alpha-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe(II), alpha-KG and dioxygen for catalysis, with the alpha-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the alpha-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe(IV)O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O(2) to generate this species is the decarboxylation of the alpha-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the alpha-keto acid to Fe(II) and the presence of a 5C Fe(II) site for the O(2) reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate alpha-KG coordination and a 5C Fe(II) site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an approximately 10(5)-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n-->pi( *) transition of the HPPD/Fe(II)/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the alpha-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO(2) is disfavored.     

Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution

Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.