Conformational rigidity of cytochrome c'-α from a thermophile is associated with slow NO binding

Abstract

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-vis absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11- 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.

Figure 1

EPR spectra of Fe(II) PhCP-α with and without NO at pH 7.5 and 10 K. Red line represents the EPR spectrum of 30 μM Fe(II) PhCP-α with 1.4 mM NO, exhibiting 16 G separated three signals by hyperfine splitting around g = 2.013, which is indicative of the 5cNO state. Blue line represents the EPR spectrum of 30 μM Fe(II) PhCP-α without NO.

Figure 2

RR spectra of Fe(II) PhCP-α at pH 8.0. (A) Fe(II) PhCP-α (407 and 442 nm excitation) obtained at room temperature and 100 K. (B) 5cNO PhCP-α (407 excitation, 100 K) prepared with ¹⁴NO (blue traces) and ¹⁵NO (red traces). Asterisks denote contributions from a minor population of the NO-free Fe(II) state, indicating that the heme faces in the ¹⁴NO sample are not fully occupied.

Figure 3

Stopped-flow kinetic analysis for PhCP-α. (A) Time-dependent absorbance shift of the reaction of 2.5 μM Fe(II) PhCP-α and 1.0 mM NO at pH 7.5 and 20°C by photodiode array detection using white light. (B) Global fitting spectra of three components of Fe(II) state (Soret peak at 425 nm), 6cNO intermediate (Soret peak at 416 nm), and 5cNO product (Soret peak at 399 nm) using the data in the presence of 1.0 mM NO. (C) NO-binding reaction scheme in PhCP-α based on the absorbance shift. The scheme is redrawn with the references for AxCP-α (14,23). (D) The representative raw data of the time-dependent absorbance shift in the reaction of 2.5 μM Fe(II) PhCP-α and 1.0 mM NO at pH 7.5 and 20°C using monochromatic light. The absorbance changes at 383, 413, and 426 nm are shown in blue, red, and gray lines, respectively. The absorbance at the start of 0.003 s is calculated as zero. (E) Plots of k_obs(A) and k_obs(B) obtained from the fitting analysis from the spectral shift at 426 nm versus NO concentration through the monochromatic light experiments. The plots indicate the means, and the error bars indicate the standard deviation based on triplicate experiments.

Figure 4

X-ray crystal structure comparison. (A) 2F_o–F_c electron density map of heme environment in the Fe(II) PhCP-α 5cNO product contoured at 1.5 σ. (B) Superposition of heme Fe and NO atoms of the 5cNO products of PhCP-α, AxCP-α, and SfCP-α. The structure colors and PDB codes used are; PhCP-α 5cNO product (purple, PDB: 8RKP), AxCP-α 5cNO product (green, PDB: 2XLM), and SfCP-α 5cNO product (cyan, PDB: 4CX9). The error values for each length are derived from the diffraction precision index (atomic position error values). (C) Superposition of the native and 5cNO states. The structure colors and PDB codes for the 5cNO products are consistent with those in (B), and those of native states used are; PhCP-α (orange, PDB: 5B3I), AxCP-α (dark green, PDB: 2YLI), and SfCP-α (pale cyan, PDB: 4ULV). (D) Comparison of the native and 5cNO states at the heme proximal region. The structure colors and PDB codes are consistent with those in (B) and (C). Yellow and black dotted lines indicate the salt bridge and hydrogen bond, respectively. Blue lines indicate the length of each atom with showing the distances (Å).

Figure 5

Thermal denaturation curves for native SfCP-α and AxCP-α. The normalized data for SfCP-α (cyan circles) and AxCP-α (green squares) are shown with fitting curves. The fitting curve for thermal denaturation of PhCP-α is reproduced from Fujii et al. (12). Sample conditions: protein concentration, 20 μM; solvent, 20 mM potassium phosphate buffer; pH, 7.0.