Identification of an intermolecular disulfide bond in barley hemoglobin.
Barley class-1 hemoglobin (Hb) and its mutated version (Cys(79) 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 approximately 10(3) 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 Cys(79) 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.
Bykova
Natalia V
NV
Department of Plant Science, University of Manitoba, Winnipeg, Man., Canada.
Igamberdiev
Abir U
AU
Ens
Werner
W
Hill
Robert D
RD
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006420:Hemeproteins / Q000032:analysis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D013329:Structure-Activity Relationship
D004220:Disulfides; C106902:HB protein, Hordeum vulgare; D006420:Hemeproteins; D010940:Plant Proteins; D003545:Cysteine