The pH Dependent Protein Structure Transitions and Related Spin-State Transition of Cytochrome c′ from Alcaligenes xylosoxidans NCIMB 11015

Abstract

The unusual magnetic/spectroscopic properties of Cytochrome c¤ (Cyt c¤) have been discussed, especially concerning the possibility of a quantum mechanically mixed-spin configuration of heme Fe(III). Here, four unique-spin species were identified from the magnetic circular dichroism (MCD) spectra of Cyt c¤ from Alcaligenes xylosoxidans (AxCyt c¤). The electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic data showed the overall conformation of AxCyt c¤ was unchanged, in complete contrast to the drastic changes in the heme MCD spectra over the range of pH 3.5 and 11.8. The pH dependency of ESI-MS, electronic absorption, MCD, and CD spectroscopic properties of AxCyt c¤ enabled us to reveal the undiscovered correlation between the protein-folding state and the electronic structure of the active site as a function of pH. The mechanism of alkaline spin-state transition through the rearrangement of the hydrogen-bonding linkage between Helix C and D is also proposed on the basis of atomic resolution crystal structure analyses (A. Takashina, M. Unno, T. Kohzuma, Chem. Lett. 2015, 44, 268).

Open-Bundle Structure as the Unfolding Intermediate of Cytochrome c’ Revealed by Small Angle Neutron Scattering

“…The SANS profile at pD 1.7 was fit well using the Debye function and gave Rg = 25.7 A, and clearly showed that the structure of Cyt c' transitioned to the unfolded random coil structure. The unfolded random coil structure of Cyt c' at pD 1.7 indicated by the SANS data is consistent with the structure previously proposed based on CD and ESI-MS spectrometry data [33]. The crystallographic structure of Cyt c' from Alcaligenes was suggested to be a dimer, but the association state in solution was unknown [42].…”

“…The Kratky plot of Cyt c' at pD 1.7 shows an intensified plateau region in the high-Q region, without a bell-shaped profile in the low-Q region (Figure 4A), strongly suggesting that Cyt c' becomes a very flexible random coil structure, in good agreement with the Debye function analysis and previous CD and ESI-MS experiments [33]. The Kratky plots of Cyt c' at pD 6.4 and 9.6 (Figure 4B and 4C) are clearly bell-shaped profiles centered at Q = 0.1 Å -1 with a plateau shape in the higher-Q region (Q > 0.2 Å -1 ), indicating a flexible moiety in the protein structure.…”

“…The spin state transition into the HS state at alkaline conditions (8 < pH < 12) is triggered by breakage of the inter-helix hydrogen-bonding linkage between helix C and helix D [29][30][31][32]. The structure of Cyt c' at pH ~13 was suggested to be a 6-coordinated low-spin (LS) state with imidazole (His120)/OHor two OHas the axial ligands, resulting in an "open-bundle" structure at pH ~13, as reported previously [33]. Very recently a program AlphaFold running on the Artificial Intelligence, Google DeepMind succeeded to provide 3D structure from the amino acid sequence [34] experimental solution structure determination of unfolded protein is very important to verify such computationally predicted protein structure.…”

“…Cyt c' was extracted from Alcaligenes xylosoxidans and purified by cation exchange chromatography (CM-Sephadex C-50, GE Healthcare) and size-exclusion chromatography (Hiload Superdex 200 pg, GE Healthcare), followed by re-crystallization according to a previous method [33]. The purity of Cyt c' was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Figure S1).…”

Stabilization of protein structure through π–π interaction in the second coordination sphere of pseudoazurin

“…Taking advantage of these detection properties, the ESI‐MS has been applied to probe both conformational change and binding/releasing the cofactor in various proteins . Recently, we reported the regulation of heme properties in cytochrome c ′ on the basis of structural information from ESI‐MS . The unfolding of azurin and amicyanin has been studied previously using ESI‐MS.…”