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On the presence of a heme‐binding domain homologous to cytochrome b 5 in Neurospora crassa assimilatory nitrate reductase
Author(s) -
Lê Kim Hô Diêp,
Lederer Florence
Publication year - 1983
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1983.tb01678.x
Subject(s) - neurospora crassa , heme , biology , biochemistry , cytochrome , cyanogen bromide , neurospora , nitrite reductase , nitrate reductase , reductase , cytochrome c , cytochrome c peroxidase , peptide sequence , cytochrome p450 reductase , mutant , stereochemistry , enzyme , coenzyme q – cytochrome c reductase , chemistry , mitochondrion , gene
Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr‐1 nit‐6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme‐binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b 5 . On SDS gels, the fraction showed two heme‐containing bands of ˜10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S‐carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b 5 starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme‐binding domain proves to be another member of the family of b 5 ‐like cytochromes.