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.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins’ roles in planta.

Globins are usually associated with oxygen carriage in vertebrates. However, plants also contain similar heme-containing proteins, called phytoglobins (Pgbs). Unlike conventional hemoglobin, these proteins are often linked to nitric oxide metabolism, energy metabolism and redox maintenance under hypoxic and related abiotic and biotic stress conditions. Class I and II non-symbiotic Pgbs (nsPgbs) have different degrees of heme hexacoordination. This involves direct interaction of the distal histidine in the E-helix with the sixth coordination site of the central iron, resulting in increased stability, in contrast to the oxygen storage linked to pentacoordinated globins, such as myoglobin (Mb). Due to their robustness, nsPgbs have substantial potential for various biomedical applications, particularly for iron supplementation. In this study, a class I nsPgb from sugar beet (Beta vulgaris ssp. vulgaris) was encapsulated in a lipid sponge phase system for potential protein delivery purposes and compared to a similar system of Mb containing nanoparticles. Bulk phases and dispersions were made with two lipid compositions (30/45/25 diglycerol monooleate (DGMO)/Capmul GMO-50/sorbitan monooleate (P80) and 28/42/30 DGMO/GMO-50/P80, where the DGMO/GMO-50 ratio was kept constant at 40/60). In addition, buffer effects on protein loading and particle formation were investigated. High concentrations of BvPgb1.2 (60 mg/mL) showed higher aggregation tendencies than Mb but these appeared to be transient. This property could be coupled to the higher isoelectric point (pI) of the BvPgb1.2 (7.85, compared to 6.8 for Mb), which make it more sensitive to small pH changes. In addition, excess protein/leakage was observed with Mb from the nanoparticles when analysed with size exclusion chromatography. This work highlighted the encapsulation efficiency of these proteins, which might be directly linked to difference in iron coordination and therefore, reactivity and lipid peroxidation. The interactions between the bulk phases and dispersion of the hemeproteins are complex, more research is needed to proper elucidate these relations in more detail, in order to facilitate the encapsulation capacity for heme-containing proteins in similar lipid-based systems.