Proteomics

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The heme regulatory motifs of heme oxygenase-2 function to transfer heme to the catalytic site for degradation


ABSTRACT: Heme regulatory motifs (HRMs) are found in a variety of proteins that are involved in diverse biological functions. In the C-terminal tail region of human heme oxygenase-2 (HO2), there are two HRMs whose cysteines form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs is independent of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the core of HO2. Using HDX-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle – when Fe3+-heme is bound to the HRMs and the core is in the apo state. The conformational changes detected are consistent with transfer of heme between binding sites. Indeed, Fe3+-heme bound to the HRMs is transferred to the apo-core upon either independently expressing the core and a construct spanning the HRM-containing tail or after single turnover of heme at the core. In addition, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We thus propose a Fe3+-heme transfer model in which heme bound to the HRMs is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John R. Engen  

LAB HEAD: John R Engen

PROVIDER: PXD017538 | Pride | 2020-03-18

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
FeMutant_10min_1.raw.zip Raw
FeMutant_10min_2.raw.zip Raw
FeMutant_10min_3.raw.zip Raw
FeMutant_10sec_1.raw.zip Raw
FeMutant_10sec_2.raw.zip Raw
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Publications

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation.

Fleischhacker Angela S AS   Gunawan Amanda L AL   Kochert Brent A BA   Liu Liu L   Wales Thomas E TE   Borowy Maelyn C MC   Engen John R JR   Ragsdale Stephen W SW  

The Journal of biological chemistry 20200309 16


Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe<sup>3+</sup>-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe  ...[more]

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