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Biochemistry ; 59(5): 704-716, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31887031

RESUMO

A defining characteristic of bacterial cytochromes (cyt's) in the P460 family is an unusual cross-link connecting the heme porphyrin to the side chain of a lysyl residue in the protein backbone. Here, via proteomics of the periplasmic fraction of the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea, we report the identification of a variant member of the P460 family that contains a methionyl residue in place of the cross-linking lysine. We formally designate this protein cytochrome "c'ß-Met" to distinguish it from other members bearing different residues at this position (e.g., cyt c'ß-Phe from the methane-oxidizing Methylococcus capsulatus Bath). As isolated, the monoheme cyt c'ß-Met is high-spin (S = 5/2). Optical spectroscopy suggests that a cross-link is absent. Hydroxylamine, the substrate for the cross-linked cyt P460 from N. europaea, did not appreciably alter the optical spectrum of cyt c'ß with up to 1000-fold excess at pH 7.5. Cyt c'ß-Met did however bind 1 equiv of H2O2, and with a slight excess, Mössbauer spectroscopy indicated the formation of a semistable ferryl (FeIV═O) Compound II-like species. The corresponding electron paramagnetic resonance showed a very low intensity signal indicative of a radical at g = 2.0. Furthermore, cyt c'ß-Met exhibited guaiacol-dependent peroxidase activity (kcat = 20.0 ± 1.2 s-1; KM = 2.6 ± 0.4 mM). Unlike cyt c'ß-Met, cyt P460 showed evidence of heme inactivation in the presence of 2 equiv of H2O2 with no appreciable guaiacol-dependent peroxidase activity. Mutagenesis of the cross-linking lysyl residue to an alanine in cyt P460, however, reversed this lack of activity.


Assuntos
Citocromos c/metabolismo , Heme/metabolismo , Compostos de Ferro/metabolismo , Lisina/metabolismo , Nitrosomonas/química , Peroxidase/metabolismo , Citocromos c/química , Citocromos c/genética , Espectroscopia de Ressonância de Spin Eletrônica , Heme/química , Compostos de Ferro/química , Lisina/química , Modelos Moleculares , Nitrosomonas/citologia , Nitrosomonas/metabolismo , Peroxidase/química , Proteômica , Espectroscopia de Mossbauer
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