Conferment of Folding Ability to a Naturally Unfolded Apocytochrome c through Introduction of Hydrophobic Amino Acid Residues

Abstract

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…Proteins in the cytochrome c (cyt c ) protein family have been studied extensively in terms of protein folding and stability . Met80 and His18 are coordinated to the heme iron in cyt c , creating a relatively high redox potential for electron transfer.…”

“…AA cyt c 555 is globular as other cyt c proteins, but possesses an exceptionally long, extra 3 10 ‐α‐3 10 helix containing heme‐ligating Met61 . The high thermostability of AA cyt c 555 has been ascribed to the possession of the extra 3 10 ‐α‐3 10 helix and tight packing of its hydrophobic residues inside the protein …”

“…18,19 Proteins in the cytochrome c (cyt c) protein family have been studied extensively in terms of protein folding and stability. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Met80 and His18 are coordinated to the heme iron in cyt c, creating a relatively high redox potential for electron transfer. The redox potential of M80A cyt c was lower than that of wildtype cyt c, due to the loss of the Met80-heme iron coordination bond, 36 whereas M80A cyt c exhibited unusual O 2 and CO binding properties.…”

“…29 The high thermostability of AA cyt c 555 has been ascribed to the possession of the extra 3 10 -a-3 10 helix and tight packing of its hydrophobic residues inside the protein. 29,33 Domain swapping is a protein oligomerization mechanism, which has been suggested as a general property of all proteins. [38][39][40][41][42][43][44][45][46][47][48] It has been observed for many proteins, including proteins which may form amyloid fibrils.…”

Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

“…However, many aspects of c-type cytochromes are still to be unveiled, from the control and fine-tuning of electron transfer reactions and heme reactivity [1–3] to the description of Cyt c folding pathways and stability [4–6]. The presence of the covalently bound heme prosthetic group dictates the functions of Cyts c, which are associated mainly with electron transfer processes in aerobic and anaerobic respiration and in photosynthesis [7, 8]; however, it is now clear that Cyts c play important roles also in other cellular processes such as H 2 O 2 scavenging, cytochrome c oxidase assembly [9], lipid signaling [10], or apoptotic processes in the eukaryotic cells [11, 12].…”