The Cytochrome c Fold Can Be Attained from a Compact Apo State by Occupancy of a Nascent Heme Binding Site

Abstract

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c 552 from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains ϳ14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R h , 16.6 Å). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c 552 with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c 552 from the apo state on the addition of heme has been demonstrated using NMR techniques.

In Vitro Studies on Thioether Bond Formation between Hydrogenobacter thermophilus Apocytochrome c552 with Metalloprotoporphyrin Derivatives

“…This is evidence that the PPIX without a metal is capable of binding non-covalently to the apocytochrome. This observation is in agreement with the report that 8-anilino-1-naphthalenesulfonate binds to a prefolded state of the apoprotein (27). Observations that hydrophobic molecules can interact with apocytochrome from horse heart have been described previously (28).…”

An Obligatory Intermediate in the Folding Pathway of Cytochromec552 from Hydrogenobacterthermophilus

“…The difference in their thermodynamic stabilities (⌬⌬G ϳ 2.9 kcal mol Ϫ1 at pH 4.7) suggests that, for H. thermophilus cytochrome c 552 , the stabilization of the native state and of the partially folded intermediate may be related to the same characteristic structural features. In this respect, it is interesting that, contrary to what was observed for mitochondrial cytochrome c (32), also the apoform of this thermophilic cytochrome c is surprisingly stable and partially folded; indeed, apocytochrome c 552 is able to bind the heme group in vitro, suggesting that it may contain a nascent heme binding site (33,34).…”

The Escherichia coli Cytochrome cMaturation (Ccm) System Does Not Detectably Attach Heme to Single Cysteine Variants of an Apocytochrome c

“…However, our data indicate that there is competition between the rate of heme binding to apoprotein in the cytoplasm and the rate of apoprotein translocation to the periplasm by the general type II secretion (Sec) proteins. Translocation may be slowed because H. thermophilus apocytochromes c 552 are quite structured (36), and the Sec system transports unfolded proteins (37).…”