Characterization of a globin-coupled oxygen sensor with a gene-regulating function.
Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate.
Thijs
Liesbet
L
Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Antwerp, Belgium.
Vinck
Evi
E
Bolli
Alessandro
A
Trandafir
Florin
F
Wan
Xuehua
X
Hoogewijs
David
D
Coletta
Massimiliano
M
Fago
Angela
A
Weber
Roy E
RE
Van Doorslaer
Sabine
S
Ascenzi
Paolo
P
Alam
Maqsudul
M
Moens
Luc
L
Dewilde
Sylvia
S
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry / Q000502:physiology*; D006639:Histidine / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013059:Spectrum Analysis, Raman; D013997:Time Factors
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D006639:Histidine; D007501:Iron; D010100:Oxygen