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. 2021 Dec 6;6(50):34912-34919.
doi: 10.1021/acsomega.1c05564. eCollection 2021 Dec 21.

Characterization of a Cobalt-Substituted Globin-Coupled Oxygen Sensor Histidine Kinase from Anaeromyxobacter sp. Fw109-5: Insights into Catalytic Regulation by Its Heme Coordination Structure

Kenichi Kitanishi  1 , Motoyuki Shimonaka  1 , Masaki Unno  2   3
Affiliations

Characterization of a Cobalt-Substituted Globin-Coupled Oxygen Sensor Histidine Kinase from Anaeromyxobacter sp. Fw109-5: Insights into Catalytic Regulation by Its Heme Coordination Structure

Kenichi Kitanishi et al. ACS Omega. .

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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Purification of Fe-AfGcHK and Co-AfGcHK. (A) Purity of Fe-AfGcHK and Co-AfGcHK determined by SDS-PAGE analysis using 12% gels. Molecular mass markers, denoted by M, are shown in the left lane. Lane 1, Fe-AfGcHK; lane 2, Co-AfGcHK. (B) Elution profiles of Fe-AfGcHK and Co-AfGcHK on a gel filtration column reveal that both proteins behave as dimers. Molecular mass markers are shown at the top.
Figure 2
Figure 2
Far-UV CD spectra of Fe-AfGcHK (red line) and Co-AfGcHK (blue line). Protein concentration was 20 μM, and the buffer was 20 mM Tris-HCl, pH 8.0, 50 mM NaCl.
Figure 3
Figure 3
Absorption spectra of oxidized [Fe(III) and Co(III); top], reduced [Fe(II) and Co(II); middle], and oxygen-bound [Fe(II)-O2 and Co(II)-O2; bottom] forms of (A) Fe-AfGcHK and (B) Co-AfGcHK. Protein concentration was 4 μM, and the buffer was 50 mM Tris-HCl, pH 8.0, 100 mM NaCl. The visible region of the spectrum (475–700 nm) has been enlarged 5-fold. The absorption maxima of these proteins are summarized in Table 1.
Figure 4
Figure 4
Autophosphorylation activities of Fe-AfGcHK and Co-AfGcHK. (A) Phos-tag SDS-PAGE gel patterns demonstrate a time-dependent increase in phosphorylated AfGcHK (upper band, P-HK) and a simultaneous decrease in nonphosphorylated AfGcHK (lower band, HK) catalyzed by the various complexes of Fe-AfGcHK and Co-AfGcHK. Data were obtained at the indicated times after initiation of the reaction. (B, C) Time-courses of autophosphorylation of (B) Fe-AfGcHK and (C) Co-AfGcHK for oxidized [M(III); open triangles], reduced [M(II); closed circles], and oxygen-bound [M(II)-O2; open circles] forms. M = Fe or Co. Data are presented as means ± S.D. of at least three independent experiments.
Figure 5
Figure 5
Proposed coordination structures of heme and cobalt porphyrin relevant to the catalytic activities of Fe-AfGcHK and Co-AfGcHK, respectively. The 6cLS complexes [Fe(III), Fe(II)-O2, Co(III), and Co(II)-O2] are fully active forms, the 5cHS complex [Fe(II)] is inactive, and the 5cLS complex [Co(II)] is moderately active, the latter of which is a novel finding of this study. By analogy with the Fe(II)-O2 complex of Fe-AfGcHK, Tyr45-OH is predicted to interact with the proximal O atom, but interaction(s) with the distal O atom cannot be totally ruled out for the Co(II)-O2 complex of Co-AfGcHK. Color codes are similar to those of traffic lights, with fully active shown in green, moderately active in yellow, and inactive in red.
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