Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Abstract

Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.

Comparison of DFT and ab initio QM/MM methods for modelling reaction in chorismate synthase

“…FMNH − is stabilized in the active site of the enzyme by hydrogen bonds with Arg45. These hydrogen bonds are most stabilizing for the reduced form FMNH − , FMNH − can exist in the active site without a discrete reaction step for its formation, the protonation of Asp339 is not required to stabilize it [13]. Arg45 may be a more suitable candidate for a base to abstract the proton from FMNH 2 .…”

“…FMN has been shown to bind to apo-CS in the reduced form [12] but it is thought to become protonated by His110 due to conformational changes after EPSP binding, thereby increasing its reduction potential [8]. There is also evidence from the study of flavin-dependent α-hydroxy acids that FMN could exist as FMNH − due to a shift in the pK a of the N35 position caused by surrounding residues in the active site [13]. From this information, the production mechanism for chorismate from EPSP in CS can be written in several feasible ways.…”

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

“…The mechanism of substrate dehydrogenation as it is now understood is presented in Figure 1. In view of the sequence similarities between members of the family of a-hydroxy acid-oxidizing enzymes (LC & and of the structural similarity between flavocytochrome b2 and glycolate oxidase (Lindqvist et al, 1991), it appears reasonable to assume that this mechanism applies in broad outline to all family members (Ghisla & Massey, 1989;Lederer, 1992). Figure 1 indicates that the active site base that captures the substrate aproton is His 373.…”

“…Whatever the actual pK, of His 373, the fact is that when this residue is under its acid form in the reduced enzyme, its proton appears to be very sticky, since a freely accessible, normally ionizing histidine would be expected to exchange its proton at a rate Lederer, 1991aLederer, , 1992. In panel B, the arrow from the substrate a-carbanion to flavin NS only indicates that electrons leave the substrate and enter the cofactor at N5; it is not meant to indicate the way they do this (for a discussion concerning this point, see Lederer, 1991a).…”

“…Therefore, it would be interesting to understand the reasons for this stickiness. Several possible structural factors were invoked in this respect (Lederer, 1991a(Lederer, , 1992. The 3D structure of flavocytochrome bZ shows 2 states of the active site, owing to positional disorder of the heme-binding domain (Xia et al, 1987;Xia & Mathews, 1990).…”