PMID- 16342963
OWN - NLM
STAT- MEDLINE
DCOM- 20060323
LR  - 20181113
IS  - 0006-2960 (Print)
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Linking)
VI  - 44
IP  - 50
DP  - 2005 Dec 20
TI  - Spectroscopic and biochemical characterization of heme binding to yeast Dap1p and 
      mouse PGRMC1p.
PG  - 16729-36
AB  - Yeast damage-associated response protein (Dap1p) and mouse progesterone receptor 
      membrane component-1 protein (mPGRMC1p) belong to a highly conserved class of 
      putative membrane-associated progesterone binding proteins (MAPR), with Dap1p and 
      inner zone antigen (IZA), the rat homologue of mPGRMC1p, recently being reported 
      to bind heme. While primary structure analysis reveals similarities to the 
      cytochrome b(5) motif, neither of the two axial histidines responsible for 
      ligation to the heme is present in any of the MAPR proteins. In this paper, EPR, 
      MCD, CD, UV-vis, and general biochemical methods have been used to characterize 
      the nature of heme binding in both Dap1p and a His-tagged, membrane 
      anchor-truncated mPGRMC1p. As isolated, Dap1p is a tetramer which can be 
      converted to a dimer upon addition of 150 mM salt. The heme is noncovalently 
      attached, with a maximal, in vitro, heme loading of approximately 30%, for both 
      proteins. CD and fluorescence spectroscopies indicate a well-ordered structure, 
      suggesting the low level of heme loading is probably not due to improperly folded 
      protein. EPR confirmed a five-coordinate, high-spin, ferric resting state for 
      both proteins, indicating one axial amino acid ligand, in contrast to the 
      six-coordinate, low-spin, ferric state of cytochrome b(5). The MCD spectrum 
      confirmed this conclusion for Dap1p and indicated the axial ligand is most likely 
      a tyrosine and not a histidine, or a cysteine; however, an aspartic acid residue 
      could not be conclusively ruled out. Potential axial ligands, which are conserved 
      in all MAPRs, were mutated (Y78F, D118A, and Y138F) and purified to homogeneity. 
      The Y78F and D118A mutants were found to bind heme; however, Y138F did not. This 
      result is consistent with the MCD data and indicates that Tyr138 is most likely 
      the axial ligand to the heme in Dap1p.
FAU - Ghosh, Kaushik
AU  - Ghosh K
AD  - Department of Chemistry and Biochemistry, University of California, Santa Cruz, 
      California 95064, USA.
FAU - Thompson, Alisha M
AU  - Thompson AM
FAU - Goldbeck, Robert A
AU  - Goldbeck RA
FAU - Shi, Xiaoli
AU  - Shi X
FAU - Whitman, Stephanie
AU  - Whitman S
FAU - Oh, Eric
AU  - Oh E
FAU - Zhiwu, Zhu
AU  - Zhiwu Z
FAU - Vulpe, Chris
AU  - Vulpe C
FAU - Holman, Theodore R
AU  - Holman TR
LA  - eng
GR  - R01 GM056062/GM/NIGMS NIH HHS/United States
GR  - EB02056/EB/NIBIB NIH HHS/United States
GR  - DBI-0217922/PHS HHS/United States
GR  - R29 GM056062/GM/NIGMS NIH HHS/United States
GR  - GM56062-06/GM/NIGMS NIH HHS/United States
GR  - R01 GM056062-10/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (DAP1 protein, S cerevisiae)
RN  - 0 (Hemeproteins)
RN  - 0 (Membrane Proteins)
RN  - 0 (PGRMC1 protein, mouse)
RN  - 0 (Receptors, Progesterone)
RN  - 0 (Saccharomyces cerevisiae Proteins)
RN  - 42VZT0U6YR (Heme)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Heme/*metabolism
MH  - Hemeproteins
MH  - Membrane Proteins/chemistry/isolation & purification/*metabolism
MH  - Mice
MH  - Molecular Sequence Data
MH  - Protein Binding
MH  - Receptors, Progesterone/chemistry/isolation & purification/*metabolism
MH  - Saccharomyces cerevisiae/*metabolism
MH  - Saccharomyces cerevisiae Proteins/chemistry/isolation & purification/*metabolism
MH  - Sequence Homology, Amino Acid
MH  - Spectrum Analysis/methods
PMC - PMC2577039
MID - NIHMS60847
EDAT- 2005/12/14 09:00
MHDA- 2006/03/24 09:00
PMCR- 2008/10/31
CRDT- 2005/12/14 09:00
PHST- 2005/12/14 09:00 [pubmed]
PHST- 2006/03/24 09:00 [medline]
PHST- 2005/12/14 09:00 [entrez]
PHST- 2008/10/31 00:00 [pmc-release]
AID - 10.1021/bi0511585 [doi]
PST - ppublish
SO  - Biochemistry. 2005 Dec 20;44(50):16729-36. doi: 10.1021/bi0511585.