Conserved tryptophan in cytochrome c: importance of the unique side-chain features of the indole moiety

Abstract

The absolute conservation of tryptophan at position 59 in cytochrome c is related to the unique chemical nature of its indole moiety. The indole side chain of Trp-59 possesses three salient features: bulk, hydrophobicity and the ability of its indole nitrogen to act as a hydrogen-bond donor. Crystallographic evidence identifies the indole nitrogen of Trp-59 as having a stabilizing hydrogen-bonding interaction with the buried carboxylate group of haem propionate 7. Side-chain bulk is also likely to be important because a Phe or Leu residue can replace Trp to give an at least partly functional protein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan by using a recently developed method for stereoselective fragment religation in yeast cytochrome c. Three yeast iso-1 cytochrome c analogues were produced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylalanine, beta-(3-pyridyl)-alanine or beta-(2-naphthyl)-alanine. Replacement of Trp-59 with these non-coded amino acids allows the reasons for its conservation to be analysed, because they vary with respect to size, hydrophobicity and hydrogen-bond potential. Our results show that decreasing the bulk and hydrophobicity of the side chain at position 59 has a profound but different impact on physicochemical and biological parameters from those of abolishing hydrogen-bond donor potential. This suggests that Trp-59 has both a local and a global stability effect by solvating a buried charge and by having a key role in the packing of the cytochrome c hydrophobic core.

Multifunctional Cytochrome c: Learning New Tricks from an Old Dog

“…Thus, although these four conserved residues are not implicated in ET, their interactions are crucial for structure and stability, including preservation of the hydrophobicity of the heme pocket. Moreover, F10 has been implicated in the recognition of the HCCS enzyme, thus pointing out the importance of these residues in assembly and folding of the protein to the native state. ,,− W59 is H-bonded to one of the heme propionates, which along with the hydrophobic character of its side chain is thought to strongly modulate the electrostatic environment of the heme. , Y67 participates in an extensive redox-sensitive H-bonding network that includes a water molecule and the side chains of N52, T78, and the axial ligand M80. − ,, These interactions are regarded as important determinants of the redox potential and ET reorganization energy of cyt c . ,,− Moreover, Y67 is considered crucial in tertiary structure stabilization as it participates in H-bonds that interconnect loops 40–57 and 71–85, which are involved in pH-dependent and other conformational changes relevant to the apoptotic function. ,,− F82 is a conserved surface residue located in the region of interaction with partner redox proteins but also in close proximity to the heme and, therefore, has been implicated in modulating the redox potential and in establishing efficient interprotein ET pathways. ,, The highly conserved prolines 30, 71, and 76 are thought to play important structural roles. P30 is important in stabilizing the distal H18 ligand; P71 helps to position the proximal ligand M80 and contributes to shield the heme group from the bulk solvent. − T78, also a conserved residue, participates in a H-bonding network that is important for determining the heme environment .…”

Growth and Pigment Production on d -Tryptophan Medium by Cryptococcus gattii , Cryptococcus neoformans , and Candida albicans

“…The most likely explanation is that the intensely pigmented cells are less dense than the lighter-pigmented cells. Tryptophan is the most hydrophobic of all amino acids and has been detected along with indole derivatives in many types of cell membranes (5,12,28,37,50,57). Hence, one possible explanation for the lower density of the pigmented C. gattii cells is a higher lipid concentration.…”

Artesunate acts through cytochrome c to inhibit growth of pediatric AML cells

“…Given that artesunate induces apoptosis, we hypothesize that artesunate binding to oxidized cytochrome c could be a steric inhibitor of the phosphorylation of Y48, which would functionally promote cytochrome c dependent apoptosis. The predicted artesunate binding site is also located near W59 which is important because the nitrogen on the indole moiety interacts with the heme via hydrogen bonding and this amino acid acts to stabilize the hydrophobic core of cytochrome c 36 . If artesunate binding causes changes in the structure of cytochrome c near W59, which the UV–visible spectroscopy data indicates, then it could also destabilize the hydrogen bonds between W59 and the heme group resulting in the observed spectral shifts in features of the heme moiety, Fig.…”

“…2 . In addition to the role W59 plays in stabilizing the heme group through hydrogen bonding it also plays a role in the stability of the hydrophobic core of the cytochrome c structure and artesunate binding could functionally destabilize the global structure of oxidized cytochrome c likely altering its function in electron transport 36 .…”