MECHANISM OF ONE-ELECTRON OXIDATION OF NAD(P)H AND FUNCTION OF NADPH BOUND TO CATALASE

Biologically important 1e- oxidations of NAD(P)H are rare. Catalase compound II and Fe(CN)63- are both strong obligate 1 e- oxidants. Using N-methyl-1,10-dihydroacridan (MAH) and N-methyl-1,10-dideuterioacridan (MAD) as models for N-alkyl- 1,4-dihydronicotinamide and N-alkyl- 1,4-dideuterionicotinamide, the mechanism for oxidation has been shown to involve (i) 1e- oxidation by Fe(CN)63- to yield the radical cation MAH+.; (ii) general-base-catalyzed proton abstraction from C10-H(D) of MAH+. to provide the neutral radical MA.; and (iii) rapid 1e- oxidation of MA. by Fe(CN)63- to provide the product N-methylacridinium cation (MA+) (Scheme I). Rate constants for general-base catalysis of MAH+. --> MA. by H2O, formate, acetate and imidazole are associated with deuterium kinetic isotope effects (k(MAH)/k(MAD)) of approximately 7-10, 4.6, 5.4, and 5.6, respectively (Bronsted beta value of 0.2). The magnitudes of the deuterium isotope effects and beta when proton abstraction is rate limiting suggest the transition state for the proton transfer to involve tunneling and to be early. With the X-ray structure of bovine catalase (Rossmann et al, ref 5), the imidazole bases of His234 and His304 in the NADPH pocket are identified as possible general-base catalysts for NADPH+. --> NADP. in the sequential -1e-, -1H+, -1e- oxidations of the NADPH moiety. Calculations of electron-tunneling pathways between NADPH (14DHN) and hypervalent iron protoporphyrin-IX (PP-IX) species were performed using PATHWAYS II (version 2.01). By use of X-ray and dynamics simulated structures, two major paths were identified: A, 14DHN-jump-Pro150-Thr149-jump-Asn147-jump-vinyl CH2=CH- [PP-IX pyrrole ring C]-Fe, and B, 14DHN-jump-Pro150-Thr149-Hbond-Ser216-jump-CH2=CH-[PP-IX pyrrole ring C]-Fe. Both paths involve short through-space jumps in addition to sigma-tunnels. Pathway B also utilizes an Hbond between Thr149(C=O) and Ser216(O-H). In the X-ray structure paths A and B have nearly identical coupling efficiencies with the PP-IX ring and iron atom, but the dynamics simulated structure favors path B by 10-fold. The favoring of path B is the result of movement of the Asn147 sidechain away from PP-IX, along with shortening of the Thr149(C=O)-(HO)Ser216 interstrand hydrogen bond during MD. The calculated coupling elements associated with paths A and B were within 1 order of magnitude of the other, indicating that both pathways contribute substantially to the overall tunneling effect. Neither axial ligand on the iron (proximal Tyr357 or distal H2O) takes part in electron transfers by the PATHWAYS II analysis. Conformational features (torsion angles X(n) and X(am) and puckering angles alpha(C) and alpha(N), Chart I) of the 1,4-dihydronicotinamide (14DHN) nucleotide were analyzed from the dynamics structures. The average value of X(n) of 96-degrees dictates a syn conformation for the 14DHN nucleotide, with the pro-S hydrogen (H(S)) on C4 of 14DHN pointed in the direction of iron protoporphyrin-IX (PP-IX). X(am) values observed during MD (between 126 and 174-degrees) indicate that the C=O of the CONH2 remains trans to C2 (Chart III) and on the B-face adjacent to H(S) throughout MD. The puckering angles alpha(C) and alpha(N) (Chart I) have average values of 7 and 13-degrees, respectively, where N1 and C4 of 14DHN are displaced out of the plane of the ring to a quasi-boat form. Anisotropy of the quasi-boat conformation points the ''bow and stern'' of the quasi-boat (C4 and N1) toward the PP-IX with H(S) pseudo-axial. It is suggested that NADPH in catalase acts not only as a rescuer of inactive catalase (in the compound II state) but also as a fuse for active catalase (in the compound I state) in the presence of very low concentrations of H2O2.