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.

Graphical abstract

Fig. 1

Left side: Fno catalyzed reaction. Fno catalyzes the reversible reduction of NADP⁺. The pro-S hydride on carbon 5 of F₄₂₀H₂ (shown in red) is transferred to carbon 4 of NADP⁺, producing NADPH. Right side: R represents the side chain of the F₄₂₀ cofactor and its precursors, which are structurally separated by the dashed lines. The structural side chain for FO includes the ribitol moiety of the R group. The structural side chain for F⁺, includes the ribitol and the phosphate moieties of the R group. The structural side chain for F₄₂₀-0, includes the ribitol, phosphate and lactyl moieties of the R group. Finally, the F₄₂₀-1 structural motif includes the entire R-side chain (the number 1 represents the length of the polyglutamate tail) , , , . R′ represents the NADPH side chain. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 2

Crystal structure of Fno. A: homodimeric quaternary structure of Fno, in the presence of oxidized F₄₂₀ cofactor and NADP⁺. B: active site of Fno, PDB file 1jax . The C5 of F₄₂₀ and C4 of NADP⁺ are 3.1 Å apart, positioned for a direct hydride transfer. I135 is positioned on the NADP⁺ side, with a 3.1 Å distance from NADP⁺ within the crystal structure.

Fig. 3

Sequence alignment of Fno from various sources. Conserved amino acids are shown in red, green are strongly similar amino acids, blue are weakly similar, while black are not conserved. Note: I135 (shown in bold) is conserved. The online program, Clustal ω was utilized to create the amino acid sequence alignment presented here. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 4

The steady state double-reciprocal plots for wtFno (A), I135V Fno (B), I135A Fno (C) and I135G Fno (D) by varying NADPH concentrations. The reaction is carried out with 25 µM FO and 0.2  μM Fno in 50 mM MES/NaOH (pH 6.5) buffer at 22 °C. These plots were made by plotting 1/k vs. 1/[NADPH], displaying a concave downward curvature, which indicates negative cooperativity .

Fig. 5

The Fno pre steady-state traces at 420 nm. The hydride is transferred to FO from NADPH by Fno. Each trace represents varying Fno concentrations: 1.0 μM Fno (solid circles), 1.5 μM Fno (open circles), and 2.0 μM Fno (solid triangles). The plots were fitted to Eq. (5) and represent the three Fno variants as follows: A (I135V Fno), B (I135A Fno), and C (I135G Fno). The reactions were carried out in 50 mM MES/NaOH (pH 6.5) buffer at 22 °C. Fno was mixed with 10 μM NADPH, forming the Fno-NADPH complex. FO (25 μM) in 50 mM MES/NaOH, pH 6.5 was then mixed with the Fno-NADPH complex. The detailed calculation of kinetic parameters and plot fitting of these graphs along with wtFno graphs are shown in the supplemental information.

Fig. 6

Diagram showing possible connection between methanogenic intermediates and glycolytic intermediates within Fno producing cells. The glycolytic intermediates are shown with potential input from F₄₂₀H₂ and NADPH produced from Fno . Enzymes connecting the pathways include: Fdh, formate dehydrogenase, glyceraldehyde-3-phosphate (G3P): ferredoxin oxidoreductase (GAPOR), G3P dehydrogenase (GAPDH), and phosphoglycerate kinase (PKG), an ATP-dependent enzyme .