Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG

Abstract

The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)═O moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the Fe(IV)═O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-Fe(IV) redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-Fe(IV) state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the hemes.

Figure 1

MauG-catalyzed biosynthesis of TTQ from preMADH. The post-translational modifications that are catalyzed by MauG are colored red.

Figure 2

Comparison of the absorption spectra of diferric forms of WT with those of variant MauGs. (A) Overlay of the spectra of 3.5 μM diferric WT MauG (black) and Q103E MauG (blue). (B) Overlay of the spectra of 3.1 μM diferric WT MauG (black) and diferric Q103A MauG (green). (C) Overlay of the spectra of 3.5 μM diferric WT MauG (black) and Q103N MauG (red).

Figure 3

Comparison of the absorption spectra of oxidized and reduced forms of WT and Q103 variant MauGs. (A) Overlay of the spectra of 3.5 μM diferric WT MauG before (—) and after (---) reduction by sodium dithionite. (B) Overlay of the spectra of 3.5 μM diferric Q103N MauG before (—) and after (---) reduction by sodium dithionite. (C) Overlay of the spectra of 3.0 μM diferric Q103E MauG before (—) and after (---) reduction by sodium dithionite. (D) Overlay of the spectra of 3.5 μM diferric Q103A MauG before (—) and after (---) reduction by sodium dithionite.

Figure 4

(A) Electron density for the high-spin heme and Q103N mutation site in MauG from the Q103N MauG–preMADH crystal structure, showing difference density for a model without the water molecule within hydrogen bond distance of Asn103. The 2F_o – F_c density is drawn as blue mesh contoured at 1.0σ. Positive F_o – F_c density is drawn as green mesh contoured at 4.0σ. (B) Comparison of the Q103N (blue) and WT (PDB entry 3L4M, pink) MauG high-spin hemes and distal residues. This figure was generated using Pymol (http://www.pymol.org/).

Figure 5

Spectrochemical redox titration of Q103N MauG. The titration was performed anaerobically in 50 mM potassium phosphate (pH 7.5) at 25 °C. The fraction of MauG that was reduced was determined by comparison with the spectra of the completely oxidized and reduced forms of MauG and quantitated from the absorbance at 550 nm. The solid line is the fit of the data to eq 1.

Figure 6

Changes in absorption spectra of WT and Q103N MauG after addition of H₂O₂. Panels A–D display overlays of the Soret region (A) and NIR region (B) of the spectrum of 2.5 μM diferric WT MauG before (black line) and immediately after (red line) addition of a stoichiometric amount of H₂O₂, and of the Soret region (C) and NIR region (D) of the spectrum of 2.5 μM diferric Q103N MauG before (black line) and immediately after (red line) addition of a stoichiometric amount of H₂O₂. (E) Time course for the return to the resting spectrum of WT MauG (blue squares) and Q103N MauG (green circles) in the Soret region. This describes the transition from the red spectrum back to the black spectrum in panels A and C. The data are for the absorbance at 405 nm for WT MauG and 403 nm for Q103N MauG. The lines are the fits of each data set to a single-exponential transition. (F) Time course for the return to the resting spectrum of WT MauG (blue squares) and Q103N MauG (green circles) in the NIR region. This describes the transition from the red spectrum back to the black spectrum in panels B and D. The data are for the absorbance at 950 nm. The lines are the fits of each data set to a single-exponential transition.

Figure 7

Steady-state and single-turnover kinetic analysis. (A) Steady-state kinetics of Q103N MauG-dependent (○) and WT MauG-dependent (●) biosynthesis of TTQ from preMADH. MauG was mixed with preMADH in 0.01 M potassium phosphate buffer (pH 7.5) at 25 °C. Reactions were initiated by the addition of 100 μM H₂O₂, and the rate of appearance of quinone MADH was monitored at 440 nm. The lines are fits of the data to eq 2. (B) Single-turnover kinetics of the reaction of diferrous Q103N MauG (○) and WT MauG (●) with quinone MADH. Reaction mixtures contained a fixed concentration of diferrous MauG (1.25 μM) and varied concentrations of quinone MADH. The reaction was monitored by the decrease in absorbance at 550 nm that corresponds to the conversion of diferrous to diferric MauG. The data for the concentration dependence of the rate are fit to eq 3.