Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Deanna L. Mendez, Shalon E. Babbitt, Jeremy D. King, John D'Alessandro, Michael B. Watson, Robert E. Blankenship, Liviu M. Mirica, Robert G. Kranz

Research output: Contribution to journalArticlepeer-review


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; -69mV,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.".

Original languageEnglish (US)
Pages (from-to)2235-2240
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number9
StatePublished - Feb 28 2017
Externally publishedYes

ASJC Scopus subject areas

  • General

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