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

Abstract

The family of FMN-dependent, a-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate a-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins &flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, , J Biol Chem 260: 11 115-1 1122). This suggested the occurrence of a pK, increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and 0 4 , a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pK, drop of about half a p H unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pK, increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.

Regulation of Oxidation−Reduction Potentials through Redox-Linked Ionization in the Y98H Mutant of the Desulfovibrio vulgaris [Hildenborough] Flavodoxin: Direct Proton Nuclear Magnetic Resonance Spectroscopic Evidence for the Redox-Dependent Shift in the pKa of Histidine-98

“…Various experiments suggest that the pK a of this histidine is dramatically increased upon reduction of the FMN such that proton exchange from the imidazolium cation is very slow, leading to estimates of the pK a as high as 10 or greater. Such a large shift is very unusual and needs to be confirmed; however, it apparently has not been possible to measure its pK a directly by NMR spectroscopy because of the size of this protein (Lederer, 1992;Balme & Lederer, 1994). These observations have led to the conclusion that His373, after assisting proton abstraction from substrate, facilitates the reduction of the FMN through favorable ion-pairing interaction with the flavin hydroquinone.…”

“…Therefore, the magnitude of the pK a and midpoint potential shifts observed in the Y98H flavodoxin, while substantial, may actually be more modest than could occur within an active-site cavity. The more restricted solvent exposure of His373 in reduced flavocytochrome b 2 could explain its substantially higher pK a ; however, hydrogen bonding to Asp282 and perhaps its closer proximity to the N1/C2O region of the FMN, which carries the formal charge of the hydroquinone anion, may also contribute (Balme & Lederer, 1994). Whether these and other interactions are significant enough to raise the pK a of His373 to values greater than 10 when the flavin is reduced remains to be established.…”

Resonance Raman Study on the Oxidized and Anionic Semiquinone Forms of Flavocytochrome b2 and l-Lactate Monooxygenase. Influence of the Structure and Environment of the Isoalloxazine Ring on the Flavin Function

“…However, the increased frequency of the ν(C4dO) mode observed upon Pyr binding is indicative of a strengthening of the C4dO bond and thus of a decreased H bonding at this group. This effect may originate from either (i) a decreased H-bonding interaction between the C4dO group and the hydroxyl group of Ser 228 or (ii) the exclusion of the two water molecules out of the Pyr site (Xia & Mathews, 1990;Balme & Lederer, 1994).…”

The chemical mechanism of flavoprotein-catalysed α-hydroxy acid dehydrogenation: A mutational analysis

“…Calculating the exact value of the H-373 pK, in reduced Flb2 from experimental data requires knowing whether the proton exchange with solvent is buffer or water catalysed [42,47,49]. This has not been analysed yet.…”