Abstract
Heme-based gas sensors are an emerging class of heme proteins. AfGcHK, a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5, is an oxygen sensor enzyme in which oxygen binding to Fe(II) heme in the globin sensor domain substantially enhances its autophosphorylation activity. Here, we reconstituted AfGcHK with cobalt protoporphyrin IX (Co-AfGcHK) in place of heme (Fe-AfGcHK) and characterized the spectral and catalytic properties of the full-length proteins. Spectroscopic analyses indicated that Co(III) and Co(II)-O2 complexes were in a 6-coordinated low-spin state in Co-AfGcHK, like Fe(III) and Fe(II)-O2 complexes of Fe-AfGcHK. Although both Fe(II) and Co(II) complexes were in a 5-coordinated state, Fe(II) and Co(II) complexes were in high-spin and low-spin states, respectively. The autophosphorylation activity of Co(III) and Co(II)-O2 complexes of Co-AfGcHK was fully active, whereas that of the Co(II) complex was moderately active. This contrasts with Fe-AfGcHK, where Fe(III) and Fe(II)-O2 complexes were fully active and the Fe(II) complex was inactive. Collectively, activity data and coordination structures of Fe-AfGcHK and Co-AfGcHK indicate that all fully active forms were in a 6-coordinated low-spin state, whereas the inactive form was in a 5-coordinated high-spin state. The 5-coordinated low-spin complex was moderately active—a novel finding of this study. These results suggest that the catalytic activity of AfGcHK is regulated by its heme coordination structure, especially the spin state of its heme iron. Our study presents the first successful preparation and characterization of a cobalt-substituted globin-coupled oxygen sensor enzyme and may lead to a better understanding of the molecular mechanisms of catalytic regulation in this family.
Highlights
Heme is one of the best-known and most important cofactors required for proper biological functioning of many proteins and enzymes,[1] including myoglobin, hemoglobin, cytochrome c, cytochrome P450, and nitric oxide synthase, among others.[1−4]Heme functions as the site for sensing gaseous molecules, including O2, NO, and CO, in heme-based gas sensor proteins.[3−6] Generally, heme-based gas sensor proteins are composed of a heme-bound gas sensor domain at the Nterminus and a functional domain at the C-terminus
Af GcHK was expressed in Escherichia coli, reconstituted with heme by adding heme to the crude extract after disrupting E. coli cells by sonication, and purified as hemebound form.[10,12]
The metal contents of purified Fe-Af GcHK and Co-Af GcHK were quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES)
Summary
Heme (iron protoporphyrin IX) is one of the best-known and most important cofactors required for proper biological functioning of many proteins and enzymes,[1] including myoglobin (oxygen storage), hemoglobin (oxygen transfer), cytochrome c (electron transfer), cytochrome P450, and nitric oxide synthase (oxygen activation), among others.[1−4]Heme functions as the site for sensing gaseous molecules, including O2, NO, and CO, in heme-based gas sensor proteins.[3−6] Generally, heme-based gas sensor proteins are composed of a heme-bound gas sensor domain at the Nterminus and a functional domain at the C-terminus. Heme (iron protoporphyrin IX) is one of the best-known and most important cofactors required for proper biological functioning of many proteins and enzymes,[1] including myoglobin (oxygen storage), hemoglobin (oxygen transfer), cytochrome c (electron transfer), cytochrome P450, and nitric oxide synthase (oxygen activation), among others.[1−4]. Association/dissociation of gaseous molecules to/from the heme iron induces structural changes in the sensor domain. These structural changes are transduced to the functional domain, thereby switching on/off transcription or catalytic reactions.[3−7] Globin-coupled oxygen sensors constitute an important family of oxygen sensor proteins in which the heme-. Af GcHK consists of an N-terminal hemebound globin sensor domain and a C-terminal histidine kinase domain. Increasing numbers of genes encoding orthologous proteins are being found in many bacterial genomes
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