A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Neuroglobin (Ngb) is a cytosolic heme protein that plays an important role in protecting cells from apoptosis through interaction with oxidized cytochrome c (Cyt c) released from mitochondria. The interaction of reduced Ngb and oxidized Cyt c is accompanied by electron transfer between them and the reduction in Cyt c. Despite the growing number of studies on Ngb, the mechanism of interaction between Ngb and Cyt c is still unclear. Using Raman spectroscopy, we studied the effect of charged amino acid substitutions in Ngb and Cyt c on the conformation of their hemes. It has been shown that Ngb mutants E60K, K67E, K95E and E60K/E87K demonstrate changed heme conformations with the lower probability of the heme planar conformation compared to wild-type Ngb. Moreover, oxidized Cyt c mutants K25E, K72E and K25E/K72E demonstrate the decrease in the probability of methyl-radicals vibrations, indicating the higher rigidity of the protein microenvironment. It is possible that these changes can affect electron transfer between Ngb and Cyt c.

By using Raman spectroscopy and scanning ion-conductance microscopy, the researchers studied the morphology and state of molecules in rat brain neurons and astrocytes induced from pluripotent stem cells of healthy donors and patients with hereditary Parkinson’s disease. The researchers established that typical bands of Raman and surface-enhanced Raman spectra of neurons and astrocytes allowed studying the distribution and conformation of a series of biological molecules (proteins, lipids, cytochromes) in healthy and unhealthy states. It was shown that in Parkinson’s disease, there was a decrease in the protein content and an increase in the proportion of reduced cytochromes in the respiratory chain of astrocyte mitochondria. When comparing the morphology of astrocyte bodies and processes, it was established that the height and cross-sectional area of astrocyte processes obtained from cells of patients with hereditary Parkinson’s disease were significantly greater than in healthy patients. The developed approach to recording the distribution and conformation of molecules in neurons and astrocytes, as well as to studying the morphology of astrocyte processes allows diagnosing the functional state of cells and investigating the mechanism of the Parkinson’s disease pathogenesis.

Interference Microscopy for Cellular Studies: 10.4018/978-1-60566-076-9.ch037: This chapter describes the application of interference microscopy and double-wavelet analysis to noninvasive study of cell structure and function. We present