Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Abstract

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c. In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli. Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; −19 mV, M81A; −69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these “release mutants.”

Biosynthesis of Single Thioether c-Type Cytochromes Provides Insight into Mechanisms Intrinsic to Holocytochrome c Synthase (HCCS)

“…Accordingly, both C18A and C18S cyt c variants co-expressed with WT HCCS yielded very low levels of released, folded cyt c (<3% WT cyt c). 22 Intensity of the heme stain signal from the Cys18 cyt c variants produced by WT HCCS effectively reflect the substantial disparity in protein yields obtained during purification when compared to WT cyt c yields (Figure 5A, compare lanes 3 and 5 with lane 1). This result indicates that both alanine and serine substitutions exhibit similar low levels of release from HCCS (step 4).…”

“…12, 22 For higher purity, a polyhistidine tag (6x-His) was added to the C-terminus for a cobalt-affinity chromatography step (Figure 2A). Single thioether cyt c variants were constructed at the Cys15 position ( i.e.…”

“…To obtain adequate levels of released Cys18 cyt c variants to study, we recently developed the use of selected HCCS “release mutants.” 22 One of these HCCS release mutants has an alanine substitution at the conserved Glu159 residue of HCCS, (which is changed to a lysine in the genetic disorder microphthalmia with linear skin defects (MLS) 34 ). The E159A HCCS release mutant facilitates the higher synthesis of cyt c variants with substitutions at axial ligand His19 and at Cys18.…”

“…11 Using the E159A HCCS “release mutant” to produce the C18A/S cyt c variants effectively shifted this balance towards product release, thus facilitating biosynthesis and purification of C18A/S cyt c for analysis. 22 …”

“…11 We very recently have shown that this HCCS “release mutant” biosynthesizes sufficient levels of CXXXH ( i.e. C18A/S) cyt c variants 22 for the biochemical analysis described here. Because redox potential and stability of a C18S cyt c variant are similar to WT, there does not appear to be selection against this variant.…”