RGD Reference Report - Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands. - Rat Genome Database

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Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands.

Authors: Davis, CA  Dhawan, IK  Johnson, MK  Barber, MJ 
Citation: Davis CA, etal., Arch Biochem Biophys 2002 Apr 1;400(1):63-75.
RGD ID: 632262
Pubmed: PMID:11913972   (View Abstract at PubMed)
DOI: DOI:10.1006/abbi.2002.2783   (Journal Full-text)

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
NADP metabolic process  IDA 632262NADPH oxidationRGD 

Molecular Function
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
flavin adenine dinucleotide binding  IDA 632262 RGD 
heme binding  IDA 632262 RGD 
NADPH-hemoprotein reductase activity  IDA 632262 RGD 

Objects Annotated

Genes (Rattus norvegicus)
Cyb5r4  (cytochrome b5 reductase 4)

Objects referenced in this article
Gene Cyb5a cytochrome b5 type A Rattus norvegicus
Gene Cyb5r3 cytochrome b5 reductase 3 Rattus norvegicus

Additional Information