Identification of an intermolecular disulfide bond in barley hemoglobin

Barley class-1 hemoglobin (Hb) and its mutated version (Cys79 replaced by Ser) were overexpressed in Escherichia coli and purified to near homogeneity. Nano-electrospray ionization mass spectrometry (nano-ESI MS) showed that the mutated barley Hb was more readily dissociated to a monomer and was more susceptible to denaturation than the native form. The mutated Hb was oxidized to the ferric state ∼103 times faster than the non-mutated form. The increased oxidation of the mutated Hb was a result of substitution of the cysteine with a serine and not a consequence of monomer formation, per se. Tandem mass spectrometry (MS/MS) analysis revealed that Cys79 participated in intermolecular S–S bond formation. The rates of nitric oxide scavenging by non-mutated and mutated Hb were similar. We conclude that the cysteine residue is an important contributor to the quaternary and tertiary structure of barley hemoglobin. It however has no direct effect on nitric oxide-scavenging activity of barley Hb.