Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2

Abstract

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.

Keywords: autoxidation; crystallization; heme loss; hexacoordination; peroxidase activity; phytoglobin; thermal stability.

Figure 1

Size-exclusion chromatography and autoxidation measurements of BvPgb1.2 WT and Cys86Ala. (A,B) BvPgb1.2 WT and Cys86Ala, respectively. Data form 280 nm (protein backbone absorption) and 412 nm (heme-containing proteins) are presented. Both species eluted ~10.9 mL, corresponding to a molecular weight ~40 kDa (Supplementary Figure S1). (C,D) Complete autoxidation spectra for BvPgb1.2 WT 25 °C and WT at 37 °C, respectively. (E,F) Complete autoxidation spectra for BvPgb1.2 Cys86Ala 25 °C and Cys86Ala at 37 °C. For the autoxidation, a zoomed-in view is presented in the 500–600 nm range for depiction of the oxidation process from the ferrous to ferric oxidation state (Fe^II→Fe^III).

Figure 2

Crystal structure comparison of BvPgb 1.2 WT, Cys86Ala, and Barley Hb. (A) Structure comparison between BvPgb 1.2 WT (gray) and Cys86Ala (light blue), highlighting helices E and F in addition to the missing DE-loop. (B) Zoomed-in view of the heme-group, displaying the heme group, proximal and distal histidinem and the orientation of the cysteine (green) and alanine (red) in the WT and mutant, respectively. (C,D) Comparison between Cys86Ala (light blue) and Barley Hb (PDBID: 2OIF) (pink), showing orientation of the cysteine in Barley Hb (green) and BvPgb 1.2 Cys86Ala (red).

Figure 3

Thermal stability and peroxide activity measurements for BvPgb 1.2 and Cys86Ala. (A) Stability measurement for oxy- and cyanide BvPgb1.2 WT in 10× PBS at 25 °C during the 7 day incubation period. Three different transition states were detected (oxy 1, 2, and 3) for the first 2 days. The transition states for oxy 2 and 3 and cyanide was detected throughout the incubation period. (B) Stability measurement for oxy-BvPgb1.2 Cys86Ala in 1× and 10× PBS at 25 °C during the 7 day incubation period. (C) Stability measurement for cyanide- and oxy-Cys86Ala and oxy-BvPgb1.2 in 10× PBS at 37 °C during the 7 day incubation period. (D,E) Peroxidase activity for WT and Cys86Ala at 22 °C and 37 °C, respectively. The measurements were conducted in triplicate (n = 3), and statistical significance (p < 0.05) was determined using an unpaired t-test.

Figure 4

Heme-loss assay using ApoMb as a heme-scavenger. (A,B) Fetal hemoglobin (HbF) blank sample and addition of ApoMb, respectively. The blank sample retained the initial spectrum after 20 h, while the Mb spectrum was detected in the right panel, most predominately seen as an increase in absorbance at 600 nm. (C,D) BvPgb1.2 WT blank sample and addition of ApoMb, respectively. In both cases, the initial spectra remained constant, i.e., no Mb spectrum at 600 nm could be detected. (E,F) BvPgb1.2 Cys86Ala blank sample and addition of ApoMb, respectively. In both cases, the initial spectra remained constant, i.e., no Mb spectrum at 600 nm could be detected. All experiments were conducted at 25 °C; spectra were measured in 10 min intervals for 20 h, and the ferric form of the Hbs was used.