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- Title
Modulation of NO binding to cytochrome c′ by distal and proximal haem pocket residues.
- Authors
Barbieri, Sonia; Murphy, Loretta M.; Sawers, R. Gary; Eady, Robert R.; Hasnain, S. Samar
- Abstract
We have cloned and expressed the cycP gene encoding cytochrome c′ from Alcaligenes xylosoxidans and generated mutations in Arg-124 and Phe-59, residues close to the haem, to probe their involvement in modulating the unusual spin-state equilibrium of the haem Fe and the unique proximal mode of binding of NO to form a stable five-coordinate adduct. Arg-124 is located in the proximal pocket of the haem and forms a hydrogen bond to the stable five-coordinated bound NO. Phe-59 provides steric hindrance at the distal face where NO binds initially to form a six-coordinate adduct. Optical spectroscopy showed altered electronic properties of the oxidised haem centre resulting from the mutations of both residues. The high affinity of the ferrous proteins for NO remained unchanged and all of the mutational variants formed a stable five-coordinate NO species ( λ Soret 395 nm) in the presence of stoichiometric concentrations of NO. However, the kinetics of the reactivity towards NO were altered, with mutation of the distal Phe-59 residue resulting in the transient six-coordinate distally bound NO adduct ( λ Soret 415 nm) not being detected. Surprisingly, substitution of the proximal residue Arg-124 with Phe, Ala, Gln or Glu also resulted in the six-coordinate adduct not being detected, showing that this proximal residue also modulates reactivity towards NO on the opposite haem face. In contrast, the R124L substitution retained the property of the native protein in the initial formation of a six-coordinate NO adduct, a finding of functional importance since a Lys or an Arg residue is invariant in these proteins.
- Subjects
ALCALIGENES; GENETIC mutation; NITRIC oxide; OPTICAL spectroscopy; SPECTRUM analysis
- Publication
Journal of Biological Inorganic Chemistry (JBIC), 2008, Vol 13, Issue 4, p531
- ISSN
0949-8257
- Publication type
Article
- DOI
10.1007/s00775-008-0341-3