Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups.

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.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions. We have found that hypoxia induced an increase in H2O2 production in adult cardiomyocytes, while neonatal cardiomyocytes experienced a decrease in H2O2 levels. This finding correlates with our observation of the difference between the electron transport chain (ETC) properties and mitochondria amount in adult and neonatal cells. We demonstrated that in adult cardiomyocytes hypoxia caused the significant increase in the ETC loading with electrons compared to normoxia. On the contrary, in neonatal cardiomyocytes ETC loading with electrons was similar under both normoxic and hypoxic conditions that could be due to ETC non-functional state and the absence of the electrons transfer to O2 under normoxia. In addition to the variations in H2O2 production, we also noted consistent pH dynamics under hypoxic conditions. Notably, the pH levels exhibited a similar decrease in both cell types, thus, acidosis is a more universal cellular response to hypoxia. We also demonstrated that the amount of mitochondria and the levels of cardiac isoforms of troponin I, troponin T, myoglobin and GAPDH were significantly higher in adult cardiomyocytes compared to neonatal ones. Remarkably, we found out that under hypoxia, the levels of cardiac isoforms of troponin T, myoglobin, and GAPDH were elevated in adult cardiomyocytes, while their level in neonatal cells remained unchanged. Obtained data contribute to the understanding of the mechanisms of neonatal cardiomyocytes' resistance to hypoxia and the ability to maintain the metabolic homeostasis in contrast to adult ones.

SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential.

The balance between the mitochondrial respiratory chain activity and the cell's needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for the lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We have verified that molecule bond vibrations assessed by SERS are a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have demonstrated that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential (ΔΨ) and H+ concentration in the intermembrane space. This regulates the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cell's consumption of ATP and the overall activity. We have found that under hypertensive conditions cytochrome c heme loses its sensitivity to ΔΨ that can affect the regulation of ETC activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens broad perspectives in cell bioenergetics.

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c.

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.

Detection of Hypertension-Induced Changes in Erythrocytes by SERS Nanosensors.

Surface-enhanced Raman spectroscopy (SERS) is a promising tool that can be used in the detection of molecular changes triggered by disease development. Cardiovascular diseases (CVDs) are caused by multiple pathologies originating at the cellular level. The identification of these deteriorations can provide a better understanding of CVD mechanisms, and the monitoring of the identified molecular changes can be employed in the development of novel biosensor tools for early diagnostics. We applied plasmonic SERS nanosensors to assess changes in the properties of erythrocytes under normotensive and hypertensive conditions in the animal model. We found that spontaneous hypertension in rats leads (i) to a decrease in the erythrocyte plasma membrane fluidity and (ii) to a decrease in the mobility of the heme of the membrane-bound hemoglobin. We identified SERS parameters that can be used to detect pathological changes in the plasma membrane and submembrane region of erythrocytes.