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.
