Biophysical and structural analysis of a novel heme b iron ligation in the flavocytochrome cellobiose dehydrogenase

Frederik A.J. Rotsaert, B. Martin Hallberg, Simon De Vries, Pierre Moenne-Loccoz, Christina Divne, V. Renganathan, Michael H. Gold

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole Nδ1 and Nε2 nitrogen atoms, rather than the typical Nε2/Nε2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl Nδ1/Nε2-coordinated protoporphyrin IX iron.

Original languageEnglish (US)
Pages (from-to)33224-33231
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number35
DOIs
StatePublished - Aug 29 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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