Amorphous Aggregation of Cytochrome <i>c</i> with Inherently Low Amyloidogenicity Is Characterized by the Metastability of Supersaturation and the Phase Diagram

Despite extensive studies on the folding and function of cytochrome <i>c</i>, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome <i>c</i>, metal-bound cytochrome <i>c</i>, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome <i>c</i> was low due to high solubility, markedly unfolded apo cytochrome <i>c</i>, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome <i>c</i> increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome <i>c</i> by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome <i>c</i> variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome <i>c</i>, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome <i>c</i> is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation