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 co mpound 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 MA H+.; (ii) general-base-catalyzed proton abstraction from C10-H(D) of M AH+. 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 deut erium 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 magni tudes of the deuterium isotope effects and beta when proton abstractio n is rate limiting suggest the transition state for the proton transfe r to involve tunneling and to be early. With the X-ray structure of bo vine catalase (Rossmann et al, ref 5), the imidazole bases of His234 a nd His304 in the NADPH pocket are identified as possible general-base catalysts for NADPH+. --> NADP. in the sequential -1e-, -1H+, -1e- oxi dations of the NADPH moiety. Calculations of electron-tunneling pathwa ys 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 identifie d: A, 14DHN-jump-Pro150-Thr149-jump-Asn147-jump-vinyl CH2=CH- [PP-IX p yrrole ring C]-Fe, and B, jump-Pro150-Thr149-Hbond-Ser216-jump-CH2=CH- [PP-IX pyrrole ring C]-Fe. Both paths involve short through-space jump s in addition to sigma-tunnels. Pathway B also utilizes an Hbond betwe en Thr149(C=O) and Ser216(O-H). In the X-ray structure paths A and B h ave nearly identical coupling efficiencies with the PP-IX ring and iro n 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 sidech ain away from PP-IX, along with shortening of the Thr149(C=O)-(HO)Ser2 16 interstrand hydrogen bond during MD. The calculated coupling elemen ts associated with paths A and B were within 1 order of magnitude of t he other, indicating that both pathways contribute substantially to th e overall tunneling effect. Neither axial ligand on the iron (proximal Tyr357 or distal H2O) takes part in electron transfers by the PATHWAY S 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-dihydr onicotinamide (14DHN) nucleotide were analyzed from the dynamics struc tures. The average value of X(n) of 96-degrees dictates a syn conforma tion 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(a m) values observed during MD (between 126 and 174-degrees) indicate th at the C=O of the CONH2 remains trans to C2 (Chart III) and on the B-f ace adjacent to H(S) throughout MD. The puckering angles alpha(C) and alpha(N) (Chart I) have average values of 7 and 13-degrees, respective ly, where N1 and C4 of 14DHN are displaced out of the plane of the rin g to a quasi-boat form. Anisotropy of the quasi-boat conformation poin ts the ''bow and stern'' of the quasi-boat (C4 and N1) toward the PP-I X 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 th e presence of very low concentrations of H2O2.