%0 Journal Article
%T Conformational rigidity of thermophilic cytochrome c'-α is associated with slow binding of nitric oxide.
%A Fujii S
%A Wilson MT
%A Adams HR
%A Mikolajek H
%A Svistunenko DA
%A Smyth P
%A Andrew CR
%A Sambongi Y
%A Hough MA
%J Biophys J
%V 0
%N 0
%D 2024 Jun 26
%M 38937973
%F 3.699
%R 10.1016/j.bpj.2024.06.026
%X Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-visible absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11-fold and ∼13-fold lower, respectively. Notably, X-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.