Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

Abstract

F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased k_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^-1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.

Keywords: Dissociation constants; E. coli,, Escherichia coli; F420 cofactor; F420 cofactor, 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphoryllactyl(glutamyl)nglutamate, A. fulgidus, Archaeoglobus fulgidus; F420H2: NADP+ oxidoreductase; FO, precursor of F420 cofactor; Fno, F420H2:NADP+, oxidoreductase; Half-site reactivity; I135, Isoleucine 135; IPTG, isopropyl β-D-1-thiogalactopyranoside; Kd,, dissociation constant; Km, Michaelis-Menten constant; LB, Luria Bertani broth; NADP; NADP+, nicotinamide adenine dinucleotide phosphate; Negative cooperativity; PEI, Polyethyleneimine; Pre steady-state kinetics; Steady-state kinetics; k, rate constant; kcat, catalytic rate constant (turnover number), kcat /Km, catalytic efficiency; wtFno, wild-type Fno.