Δ 1 -Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ 1 -KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The catalytic mechanism assumes the formation of a double bond in two steps, proton abstraction by tyrosyl ion, and a rate-limiting hydride transfer to FAD. This hypothesis was never verified by quantum-mechanical studies despite contradictory results from the kinetic isotope effect (KIE) reported in 1960 by Jerussi and Ringold [Biochemistry 1965, 4 (10)]. In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4−16.3 kcal•mol −1 ) and hydride transfer (15.5−18.0 kcal•mol −1 ) are very close (1.7−2.1 kcal•mol −1 ), which explains normal KIE values for steroids labeled either at C1 or C2 atoms. We confirm that tyrosyl ion acting as the catalytic base is indeed necessary for efficient activation of the steroid. We explain the lower value of the observed KIE (1.5−3.5) by the nature of the free energy surface, the presence of diffusion limitation, and to a smaller extent, conformational changes of the enzyme upon substrate binding. Finally, we confirm the Ping-Pong bi−bi kinetics of the whole Δ 1 -dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation, and enzyme reoxidation proceed at comparable rates.
According to the current paradigm, the metal− hydroxo bond in a six-coordinate porphyrin complex is believed to be significantly less reactive in ligand substitution than the analogous metal−aqua bond, due to a much higher strength of the former bond. Here, we report kinetic studies for nitric oxide (NO) binding to a heme-protein model, acetylated microperoxidase-11 (AcMP-11), that challenge this paradigm. In the studied pH range 7.4−12.6, ferric AcMP-11 exists in three acid− base forms, assigned in the literature as 2), and [(AcMP-11)Fe III (OH)(His − )] (3). From the pH dependence of the second-order rate constant for NO binding (k on ), we determined individual rate constants characterizing forms 1−3, revealing only a ca. 10-fold decrease in the NO binding rate on going from 1 (k on(1) = 3.8 × 10 6 M −1 s −1 ) to 2 (k on (2) = 4.0 × 10 5 M −1 s −1 ) and the inertness of 3. These findings lead to the abandonment of the dissociatively activated mechanism, in which the reaction rate can be directly correlated with the Fe−OH bond energy, as the mechanistic explanation for the process with regard to 2. The reactivity of 2 is accounted for through the existence of a tautomeric equilibrium between the major [(AcMP-11)Fe III (OH)(HisH)] (2a) and minor [(AcMP-11)Fe III (H 2 O)(His − )] (2b) species, of which the second one is assigned as the NO binding target due to its labile Fe−OH 2 bond. The proposed mechanism is further substantiated by quantum-chemical calculations, which confirmed both the significant labilization of the Fe−OH 2 bond in the [(AcMP-11)Fe III (H 2 O)(His − )] tautomer and the feasibility of the tautomer formation, especially after introducing empirical corrections to the computed relative acidities of the H 2 O and HisH ligands based on the experimental pK a values. It is shown that the "effective lability" of the axial ligand (OH − /H 2 O) in 2 may be comparable to the lability of the H 2 O ligand in 1.
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