<p>The unusual magnetic/spectroscopic properties of Cytochrome <I>c</I>′ (Cyt <I>c</I>′) 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 <I>c</I>′ from <I>Alcaligenes xylosoxidans</I> (<I>Ax</I>Cyt <I>c</I>′). The ESI-MS and CD spectroscopic data showed the overall conformation of <I>Ax</I>Cyt <I>c</I>′ 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 <I>Ax</I>Cyt <I>c</I>′ 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, <I>Chem. Lett</I>. <B>2015</B>, <I>44</I>, 268).</p>