Stabilization of cytochrome b 5 by a conserved tyrosine in the secondary sphere of heme active site: A spectroscopic and computational study
Abstract Heme proteins perform a large array of biological functions, with the heme group bound non-covalently or covalently. To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins, we herein used cytochrome b 5 (Cyt b 5 ) as a model protein, and mutated Tyr30 to Phe or His by removal of Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b 5 and Y30H Cyt b 5 , as monitored by both UV–Vis and CD spectroscopy, as well as heme transfer studies from these proteins to apo-myoglobin, with wild-type Cyt b 5 under the same conditions for comparison. The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability. Moreover, we performed molecular modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b 5 was well-remained, whereas Y30H Cyt b 5 formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b 5 is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site. As demonstrated here for Cyt b 5 , it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions. Highlights The stabilization role of a conserved Tyr30 in Cyt b 5 was studied. Both Y30F Cyt b 5 and Y30H Cyt b 5 mutants exhibit reduced thermal stability. Rate constant of heme transfer to apo-Mb: Y30H Cyt b 5 >Y30F Cyt b 5 >WT Cyt b 5 . Both hydrophobic and H-bonding interactions of Tyr30 contribute to protein stability. This study provides clues for design of artificial heme proteins. Graphical Abstract [DISPLAY OMISSION]
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