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Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

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Related in: MedlinePlus

Spectral analysis reveals CO and NO binding to sAC-HD(A) Addition of an NO donor to the reduced protein shifts the maximum of the Soret peak from 425 nm (no NO, grey line) to 417 nm (with NO, black line). (B) Spectra of the reduced haem-binding domain in the presence (grey line) or absence (black line) of CO. CO shifts the Soret peak from 425 to 417 nm, the β-peak from 528 to 536 nm and the α-peak from 557 to 563 nm.
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Figure 2: Spectral analysis reveals CO and NO binding to sAC-HD(A) Addition of an NO donor to the reduced protein shifts the maximum of the Soret peak from 425 nm (no NO, grey line) to 417 nm (with NO, black line). (B) Spectra of the reduced haem-binding domain in the presence (grey line) or absence (black line) of CO. CO shifts the Soret peak from 425 to 417 nm, the β-peak from 528 to 536 nm and the α-peak from 557 to 563 nm.

Mentions: We tested NO binding to sAC-HD by adding the NO donor DEANO. DEANO shifted the Soret peak from 425 (indicative of the ligand-free reduced protein) to 417 nm, and it decreased the α- and β-bands to the point where they were no longer clearly visible (Figure 2A). In the oxidized state, the Soret maximum shifted from 418 to 410 nm upon addition of NO, and the β-peak decreased again (results not shown). The spectra thus indicate that sAC-HD can bind NO [30], independent of its oxidation state.


Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Spectral analysis reveals CO and NO binding to sAC-HD(A) Addition of an NO donor to the reduced protein shifts the maximum of the Soret peak from 425 nm (no NO, grey line) to 417 nm (with NO, black line). (B) Spectra of the reduced haem-binding domain in the presence (grey line) or absence (black line) of CO. CO shifts the Soret peak from 425 to 417 nm, the β-peak from 528 to 536 nm and the α-peak from 557 to 563 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475452&req=5

Figure 2: Spectral analysis reveals CO and NO binding to sAC-HD(A) Addition of an NO donor to the reduced protein shifts the maximum of the Soret peak from 425 nm (no NO, grey line) to 417 nm (with NO, black line). (B) Spectra of the reduced haem-binding domain in the presence (grey line) or absence (black line) of CO. CO shifts the Soret peak from 425 to 417 nm, the β-peak from 528 to 536 nm and the α-peak from 557 to 563 nm.
Mentions: We tested NO binding to sAC-HD by adding the NO donor DEANO. DEANO shifted the Soret peak from 425 (indicative of the ligand-free reduced protein) to 417 nm, and it decreased the α- and β-bands to the point where they were no longer clearly visible (Figure 2A). In the oxidized state, the Soret maximum shifted from 418 to 410 nm upon addition of NO, and the β-peak decreased again (results not shown). The spectra thus indicate that sAC-HD can bind NO [30], independent of its oxidation state.

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

Show MeSH
Related in: MedlinePlus