Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 223-240 © Pleiades Publishing, Ltd., 2024.

223

REVIEW

Mitocentricity

Dmitry B. Zorov

1,2,a

*, Polina A. Abramicheva

, Nadezda V. Andrianova

Valentina A. Babenko

1,2

, Ljubava D. Zorova

1,2

, Savva D. Zorov

1,3

, Irina B. Pevzner

1,2

Vasily A. Popkov

1,2

, Dmitry S. Semenovich

, Elmira I. Yakupova

, Denis N. Silachev

1,2

Egor Y. Plotnikov

1,2

, and Gennady T. Sukhikh

Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology, 117997 Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: zorov@belozersky.msu.ru

Received December 29, 2023

Revised January 19, 2024

Accepted January 21, 2024

Abstract— Worldwide, interest in mitochondria is constantly growing, as evidenced by scientific statistics, and

studies of the functioning of these organelles are becoming more prevalent than studies of other cellular struc-

tures. In this analytical review, mitochondria are conditionally placed in a certain cellular center, which is respon-

sible for both energy production and other non-energetic functions, without which the existence of not only the

eukaryotic cell itself, but also the entire organism is impossible. Taking into account the high multifunctionality

of mitochondria, such a fundamentally new scheme of cell functioning organization, including mitochondrial

management of processes that determine cell survival and death, may be justified. Considering that this issue is

dedicated to the memory of V. P. Skulachev, who can be called mitocentric, due to the history of his scientific activi-

ty almost entirely aimed at studying mitochondria, this work examines those aspects of mitochondrial functioning

that were directly or indirectly the focus of attention of this outstanding scientist. We list all possible known mito-

chondrial functions, including membrane potential generation, synthesis of Fe-S clusters, steroid hormones, heme,

fatty acids, and CO

. Special attention is paid to the participation of mitochondria in the formation and transport

ofwater, as a powerful biochemical cellular and mitochondrial regulator. The history of research on reactive oxy-

gen species that generate mitochondria is subject to significant analysis. In the section “Mitochondria in the center

of death”, special emphasis is placed on the analysis of what role and how mitochondria can play and determine

the program of death of an organism (phenoptosis) and the contribution made to these studies by V. P. Skulachev.

DOI: 10.1134/S0006297924020044

Keywords: mitochondria, cell, organism, phenoptosis, death, membrane potential, water, swelling, uncoupling,

, steroids, heme, fatty acids, reactive oxygen species

Abbreviations: ROS,reactive oxygen species.

* To whom correspondence should be addressed.

INTRODUCTION

This issue is dedicated to the memory of Vladi-

mir Petrovich Skulachev, who, even after his death,

was rated as the no. 1 biochemist in Russia (https://

research.com/scientists-rankings/biology-and-biochem

istry/ru). He was a biochemist not only by education,

but also by style and implementation of thinking.

His fundamental understanding of the course of bio-

chemical processes in a living cell has placed him

among the most important scientific people in the

world. Thisman has placed the study of mitochondria

at the center of his scientific existence. That is why our

work has been called “Mitocentricity”, which justifies

the actions of a great scientist who devoted his life to

the study of this unique structure.

If we begin to analyze in detail all possible met-

abolic pathways in which mitochondria participate,

then placing them in some center of biological action

ZOROV et al.224

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Distribution of publications of biomedical profile concerning the structure and function of various organelles. We note two

sharp increases in publications on mitochondria in 1988 and 1996 after publications on the first identified mutations in mito-

chondrial DNA and participation in programmed cell death, respectively. We also note the decline in publications on the cell

nucleus after the completion of the human genome project (from[2] with permission).

seems justified [1]. Given the great number of pub-

lished works on the activity of mitochondria, we limit

our consideration to their role in two polar states– life

and death. Therefore, we have divided this work into

two parts, one of which will be called “Mitochondria

at the Center of Life” and the other “Mitochondria at

the Center of Death”, of course, bearing in mind that

in the latter case we will talk about the role of mito-

chondria in the preparation and implementation of

the termination of the biological system, while the first

part, which is presented in more detail, the role of mi-

tochondria in enabling the vital activity of this system

will be presented. This scheme corresponded to the

scientific views of V. P. Skulachev, and we will analyze

some of their aspects.

We must admit that the placement of mitochon-

dria in the center of biological actions is also objec-

tive, which, in particular, is due to the fact that of all

cellular organelles, scientific interest to mitochondria

does not decrease, but rather increases, while interest

to other organelles, firstly, remains significantly low-

er, and, secondly, either it does not change much over

the years, or it shows a negative trend, as in the case

of nuclear research (figure). The figure shows that

in2022 almost 2% of all biomedical publications were

dealing with mitochondrial activity.

One can only assume that such interest is caused

by the demonstration of the polyfunctionality of this

organelle [3]. It should be noted that throughout almost

the entire 20th century mitochondria were considered

almost exclusively as energy producers, and only their

bioenergetic function was presented in the textbooks

available at that time. The enumeration and summa-

tion of all possible (in particular, alternative to bioen-

ergetic) mitochondrial functions was largely revolu-

tionary. In terms of the evolution of the scientific view

of the role of mitochondria in the cell, there has been

a transition from the idea of the monofunctionality of

this organelle to its multifunctionality. Moreover, in

modern literature the energetic aspects of mitochon-

drial activity are being considered less and less, bring-

ing to the fore the structural and functional organi-

zation of mitochondria. It should be noted that in the

works of Vladimir Skulachev, some of which we will

discuss below, there was a transition in time from con-

sideration of purely bioenergetic aspects of mitochon-

drial activity to considering alternative mitochondrial

functions of biomedical importance.

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

It becomes clear that mitochondrial polyfunction-

ality is most likely explained by the origin of mitochon-

dria. Admittedly, mitochondria originate from certain

gram-negative bacteria, which by definition must be

multifunctional, which determines the self-sufficien-

cy necessary for survival in an environment charac-

terized by significant chemical and physical diversity

andvariability.

The transfer of bacteria into a certain eukaryotic

prototype, the internal environment of which is char-

acterized by a relatively guaranteed uniformity of com-

position and conditions, was accompanied by the loss

of a number of functions inherent in bacteria. Inpar-

ticular, there was a loss of the flagellum necessary for

movement and a number of properties uncharacteris-

tic of the bacterium were acquired (for example, the

acquisition of thermogenic function). One can only as-

sume that such interest is caused by the multifunction-

ality of this organelle [3]. Mitochondria in the process

of evolution acquired an adenine nucleotide transloca-

tor (ANT), the existence of which in bacteria would be

not only meaningless, but also fatal, because all the ATP

synthesized in the cell would be released into a dimen-

sionless space. If the point of view of the bacterial ori-

gin of mitochondria is true, then some apparently ab-

surd antagonism between mitochondria and other cell

elements becomes clear, which will be discussed later.

MITOCHONDRIA AT THE CENTER OF LIFE

Let us consider the basic, often unique, vital func-

tions inherent only in mitochondria, while we will

not dwell on those aspects that have been widely dis-

cussed in the scientific field.

It should be noted here that the bioenergetic func-

tion of mitochondria does not belong to the unique

intracellular functions, because there is anaerobic,

non-mitochondrial energy production.

Generation of the transmembrane potential

of hydrogen ions. The work of some transmembrane

enzymes may be accompanied by asymmetric charge

separation, which, for example, occurs when the

Na,KATPase located in the plasma membrane normal-

ly transfers 3Na

in exchange to 2K

[4] or the already

mentioned ANT, catalyzing the exchange of intram-

itochondrial ATP

4–

for extra-mitochondrial ADP

3–

 [5].

The same principle of charge asymmetry generation is

applicable to mitochondrial proton pumps, which cre-

ate the transmembrane potential of hydrogen ions, an

integral part of which is the electric potential on the in-

ner mitochondrial membrane (minus inside). Wehave

repeatedly pointed out the highest importance of the

mitochondrial membrane potential, which is clear-

ly not limited only to support of ATP synthesis in the

ATP synthase complex [6]. It would not be redundant

to note once again that even in critical conditions,

when the generation of membrane potential due to

the operation of proton pumps is halted (for example,

in hypoxia), the mitochondria uses either a reversed

ATP synthase system (mitochondrial ATPase as part of

complex V) [7] or coupled activation of the fumarate

reductase pathway to create the membrane poten-

tial[8, 9].

Maintenance of the membrane potential in condi-

tions unfavorable for oxidative phosphorylation, that

is, in conditions of suboptimal energetics, outwardly

seems unjustified, incomprehensible and conditionally

selfish. However, we assume that the decisive role is

played by the need to preserve the high quality of mi-

tochondria, the control of which requires a membrane

potential, with the help of which low-functional mito-

chondria are selected [10]. The criterion of this high

quality is the presence of high membrane potential

inmitochondria.

The mandatory requirement of a membrane po-

tential for the transport of proteins into the mitochon-

dria [11], which are mostly synthesized outside the

mitochondria, can also at least partially explain the

requirement of the need for a membrane potential for

the general existence of both the mitochondria and

the cell. It is significant that homeostasis of the mito-

chondrial membrane potential is a prerequisite for the

healthy existence of mitochondria and host cells, and

those mitochondria that do not meet this requirement

are subject to disposal by mitophagy [10].

The membrane potential is the driving force of the

transport of cations, in particular calcium ions, which

play an extremely important role in regulating the me-

tabolism in mitochondria and cells [6]. It is mainly due

to the accumulation of Ca

in mitochondria through

the electrogenic Ca

uniporter that mitochondria are

considered as an intracellular buffer of calcium ions

on a par with the endoplasmic reticulum [12, 13].

Quantification of the membrane potential in mito-

chondria requires various kinds of controls, while the

most widely used approach is the use of penetrating

cations, which have long been called “Skulachev ions”,

giving a credit to the founder of this approach [14, 15].

In the section related to the generation of reactive oxy-

gen species (ROS) in mitochondria, we will disclose the

role of membrane potential homeostasis in maintain-

ing ROS homeostasis, as one of the most significant dis-

coveries of Skulachev.

Synthesis of iron-sulfur clusters. Iron-sulfur

compounds are an essential component of the biologi-

cal system [16]. Perhaps they are the oldest direct pre-

cursors of life – at least so says Wächtershäuser who

hypothesized that early life was formed on the surface

of minerals containing iron sulfide [17, 18].

Iron-sulfur clusters are universal protein prosthet-

ic groups with different stoichiometric ratios of iron

ZOROV et al.226

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

and sulfur atoms and perform multiple functions in

biological systems. They are most often involved in a

variety of redox reactions, while their participation in

electron transport, ribosome biogenesis, DNA replica-

tion and repair, transcription and translation, and sev-

eral other processes is known [1]. It is assumed that

the assembly of the Fe–S cluster was an essential cause

of the emergence of mitochondria as an endosymbiont

[19, 20], since the synthesis of Fe–S clusters is neces-

sary for the survival of eukaryotic cells [21-23].

Synthesis of steroid hormones. Mitochondria

provide the synthesis of some of the most powerful

cellular regulators, namely, steroid hormones due to

the cleavage of the cholesterol side chain, which oc-

curs with the participation of one of the isoforms of

cytochrome P450 (P450ssc). This process takes place in

steroidogenic cells of the adrenal glands, gonads, pla-

centa, and brain [24]. An important role in this process

is played by the so-called a “peripheral” benzodiaze-

pine receptor (in modern classification called trypto-

phan-rich-sensory protein (TSPO), located in the outer

mitochondrial membrane, which is homologous to the

CrtK protein, a tentative oxygen sensor in the cells of

Rhodobacter bacteria [25, 26].

generation. Carbon dioxide (CO

) is formed

in the matrix of mitochondria in the Krebs cycle from

isocitrate and α-ketoglutarate, forming carbonic acid,

dissociated to form a proton and a carbonate anion.

It should be kept in mind that the higher the activity

of the Krebs cycle, the more actively the Krebs cycle is

functioning (and, correspondingly, the higher the res-

piration rate), the more CO

is formed and the greater

is the probability of acidification of the mitochondri-

al matrix (for more information, see [27]). Besides the

fact that the carbonate ion plays a very important role

in the homeostasis of cellular pH, it carries important

signaling functions [27-33].

Heme synthesis. One of the most important com-

ponents of redox reactions is heme, which is part of

hematoporphyrins and is synthesized in the mitochon-

drial matrix to provide intermediate reactions in the

cytosol [34-36].

The starting substrate of the heme synthesis cas-

cade is succinyl CoA, and the final product is pro-

toporphyrin IX, into which the ferrochelatase enzyme

introduces iron ions, completing the synthesis of iron

derivatives of heme, particular representatives of

which are cytochromes and hemo- and myoglobin.

Given the high gross content of heme iron derivatives

in higher animals, they are an important reservoir of

oxygen and iron in the organism.

Synthesis and utilization of fatty acids. In his

works, V. P. Skulachev paid considerable attention to

the relationship between mitochondria and long-chain

fatty acids. It should always be kept in mind in any

consideration of general medical issues, for example,

related to the problem of obesity, that both synthesis

and β-oxidation of fatty acids occur in mitochondria

[37]. It is also necessary to understand that the syn-

thesis of fatty acids starts with acetyl CoA, while the

synthesis competes with the non-enzymatic process

of acetylation of biological components, as a result of

which the synthesis of fatty acids to some extent pre-

vents the accumulation of acetyl CoA and, accordingly,

hyperacetylation. The same thing applies to the pro-

cess of fatty acid oxidation, the activity of which can

seriously affect the level of acetyl- and other fatty de-

rivatives of CoA and indirectly and directly affect the

acylation process, contributing to the enzymatic and

non-enzymatic modification of biological structures.

In his numerous studies, Skulachev pointed out

that the level of fatty acids in the cell will largely de-

termine the efficiency of mitochondrial energetics,

given the fact that fatty acids are uncouplers of oxida-

tive phosphorylation [38-41].

Water formation and redistribution. In this sec-

tion, we want to touch in sufficient detail a question

that is extremely rarely raised in the scientific com-

munity, namely, the issue of water formation in the

cell as one of the important regulatory mitochondrial

functions. Although mitochondrial activity is usually

judged by the level of oxygen consumption, we want

to draw attention to the fact that mitochondria con-

sume the bulk of the oxygen taken by cells, mainly as-

sociated with the formation of water and carbon diox-

ide, and this is an extremely important mitochondrial

function.

Superficially, it seems that the need to maintain

water homeostasis is not a very important problem,

but this is not so, because edema of organs (primarily

lungs or brain) is ultimately the principal cause of the

death of the organism, although edema is not a direct

consequence of mitochondrial dysfunction. Therefore,

we will review some aspects of the formation and re-

distribution of water in the cell.

Almost all the food consumed as a result of its uti-

lization by the body turns into water and carbon di-

oxide (later, when considering the functioning of mito-

chondria during detoxification, we will discuss the fate

of nitrogenous compounds, which will complement

the entire set of products formed as a result of food

consumption). So, the main product of the oxidation

of reduced equivalents as a result of the functioning

of the respiratory chain is water, which is formed in

the active center of cytochrome oxidase facing the in-

termembrane space (that is, towards the cytosol) [42].

However, it must be recognized that the calculation of

water formation solely basing on oxygen consumption

is not correct, since part of the oxygen consumed is

at least temporarily used to form oxidized lipids, pro-

teins, and nucleic acids, which is especially important

under conditions of oxidative stress.

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The reaction of water formation from 2H

formed

as a result of proton pumping and an oxygen atom

receiving two electrons from cytochrome oxidase is a

first-order reaction in oxygen. Thus, the level of the lat-

ter will determine the water flux formed in mitochon-

dria. This means that the higher the respiration rate

(coupled or uncoupled with ATP synthesis), the higher

the water production, and under uncoupled respiration,

the rate of water formation will be significantly higher

than in the coupled system. This also means that the un-

coupled oxidative phosphorylation will lead to a suffi-

ciently strong generation of water in the mitochondria,

causing the need to eliminate it from the mitochondria,

and then the cell, in order to prevent swelling of the

mitochondria and cells. On the other hand, it must be

realized that under conditions of hypoxia (physical or

chemical latter caused by inhibitors of the components

of the respiratory chain, primarily cytochrome oxidase,

for example, by carbon or nitrogen monoxides), the

rate of water formation in mitochondria due to oxida-

tion will be lower than under normoxia.

On the other hand, we admit that the chemical

reaction of ATP synthesis from ADP and P

is also as-

sociated with the formation of water, and it will be

higher with the increase of the rate of ATP synthesis,

which occurs in the active center of ATP synthase fac-

ing the mitochondrial matrix. Thus, it is easy to see

that in mitochondria, depending on internal needs and

environmental factors, processes of water formation

and utilization related to energetics occur, which re-

quire maintaining some optimal water homeostasis to

solve specific energy problems, ultimately expressed

in the problem of maintaining the ratio of delivery to

use (supply–demand problem) [43-46]. Admitting that

the main energy processes occur in the inner mem-

brane and the mitochondrial matrix, it becomes clear

that the degree of matrix hydration is crucial and is

one of the key factors in regulating the mitochondrial

energetics. It should be noted that energy-dependent

structural transitions in mitochondria, accompanied

by changes in their volume, were the subject of Sku-

lachev’s research in the 70s of the last century [47, 48].

Comparison of the electron-microscopic data with

the light scattering one using the suspension of isolat-

ed mitochondria, alternative options were suggested

for either drying of the matrix (the so-called conden-

sation observed both in state 3 according to Britton

Chance and at the first stage of uncoupling) [48, 49-53],

or its acute watering. The latter (previously we called

this condition mitochondrial edema) is called high-am-

plitude swelling [54], and it is usually an indicator of

the onset of the point of no return, as a result of in-

duction of nonspecific permeability followed by the

release of proapoptotic factors from mitochondria that

bring cell death closer [55, 56]. It is in the range be-

tween these polar states that structural changes in the

mitochondria occur in the cell, associated with either

mobilization or inhibition of energy metabolism.

The history of studying water transport in mi-

tochondria is quite long, but it cannot be considered

successful. This topic has been the subject of study by

the classics of bioenergetics, including Lehninger [57],

Green etal. [58], Hackenbrock, and others [49, 59].

The failure was largely due to the fact that mito-

chondria were recognized as imperfect osmometers

[60, 61], and the described facts of water distribution

in mitochondria did not fit into the laws of distribu-

tion in accordance with osmotic and oncotic forces.

According to the calculations of Srere [62], mito-

chondrial water can exist in a quasi-crystalline phase

and, given the very high concentration of ions, small

and large protein molecules and nucleic acids in the

cytosol or matrix of mitochondria, in principle, each

molecule, in particular a macromolecule, can be con-

sidered as a crowder, followed by the application of

the principle of molecular crowding to the state of the

water around these proteins and at a distance [63].

Calculations show a fairly large contribution of inac-

cessible water to its total concentration, which makes

it very difficult to determine available, that is, meta-

bolically active water, since considering all water as

a bulk phase is not correct. As noted by Garlid [61],

who demonstrated the distribution of uncharged sub-

stances over two phases of water in the mitochondrial

matrix, one of these phases was osmotically inactive

and had a more or less constant volume determined

by hydration, and the other was osmotically active,

and it can be called volumetric water. An osmotically

inactive compartment differs from bulk water in its

solvent properties, so that some solutes are excluded

and some are preferably dissolved.

Volumetric rearrangements occurring in mitochon-

dria with changes in physiological load change the na-

ture of molecular crowding, in particular for a well-

developed model of changes in the conformation of

nucleic acid depending on a number of environmental

factors, which include changes in the nature of hydro-

gen bonds, stacking interaction of bases, changes in

conformational entropy, changes in the concentration

of surrounding counterions and the degree of hydra-

tion [63]. In general, all these principles can be applied

to molecular crowding of protein molecules [62-66].

This must be taken into account not only to evaluate

the available water, which determines the structure

and function of proteins in a natural environment

with a high concentration of macromolecules, which

may strongly depend on dilution, but also to interpret

the data in conditions of studying the behavior of iso-

lated mitochondria in vitro with the usual use of dilute

solutions as an incubation medium. It has been report-

ed that the mitochondrial matrix contains 0.272µl/mg

of water (out of a total amount of 0.555µl/mg), which

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

does not respond to osmotic forces, which means that

almost half of the water in the matrix is bound and

osmotically inactive [67].

NMR relaxation data for a 20% protein solution

gave three different values: 10

–12

, 10

–9

, and 10

–3

, which

were explained by the existence of three types of wa-

ter (type I – water in the bulk phase; type II – bound

water; type III – non-rotating bound water) [68], and

these values can be applied to estimates of the state

of water in biological samples. While 20% of the pro-

tein contains about 90% type I water, 10% type II water

and about 0.1% type III water, a 50% protein solution

(which is close to the protein content in the mitochon-

drial matrix) contains 15-30% bound water (type II).

The two types of bound water are characterized by

ordering (structuring) depending on the distance from

the interphase, unlike bulk water, which is disordered

and in which mainly metabolic processes occur [68,

69]. All these calculations and assumptions demon-

strate the importance of even small volume changes

closely related to the percentage of metabolically ac-

tive water capable of moving in mitochondria and the

cell, as opposed to “abnormal” (using Garlid’s termi-

nology [61]), i.e., osmotically inactive, bound water.

Substances causing moderate swelling of mito-

chondria (Hoe694, Diazoxide, DUDLE, etc.) led to an

increase in the volume of mitochondria of cardiomyo-

cytes by only a few percent (up to 5), while the respira-

tion rate of these cells increased by 1/3 [70]. If we take

into account the data of Srere [71] that the volume of

water in the mitochondrial matrix is less than half of

the total volume of the matrix (the rest is occupied by

proteins), in which the proportion of metabolic water

is unknown, then even assuming that the entire pool is

metabolic water, the volume of the matrix in this case

increases by a multiple of these experimental few per-

cents, which is equivalent to a very significant change

in the volume of the matrix. If this is the case, then as

a result of such a small change in the volume of the

mitochondrial matrix, the activity of the Krebs cycle,

one of the main elements of mitochondrial bioener-

getics, may change dramatically [72-74]. This indicates

the presence of non-linear relationships between the

degree of generation and intake of water into the mito-

chondria and changes in energy production.

Several physical and related biochemical factors

specifically change with changes in the volume of the

matrix and surrounding membranes. We can name just

a few of them.

1. Changes in the compartmentalization of proteins

in the matrix, leading to the formation and disintegra-

tion of supercomplexes [67, 75-79].

2. Concentration or dilution of metabolites, cofac-

tors and inhibitors of endogenous enzymes [80, 81].

3. A change in the curvature of lipids located in

the bends of mitochondrial crystae, which leads to a

change in the oligomerization of membrane proteins

and their kinetic constants [67, 80-83].

4. Changes in the activity of protein kinases de-

pending on the volume in which they are located [84].

However, it should be noted that most calculations

of water parameters in mitochondria relate to isolated

mitochondria, the configuration of which (a strongly

expanded intermembrane space and a condensed ma-

trix) differs from the configuration of mitochondria

insitu (an expanded matrix and a narrow intracrystae

and intermembrane space), which makes calculations

of changes in the volume of the mitochondrial matrix

very difficult to carry out.

The inability to distinguish between water res-

ervoirs in the cell and mitochondria, while these res-

ervoirs are involved in transport and catalysis in dif-

ferent ways, and, on the other hand, the mismatch of

mitochondrial configurations under in vitro and in situ

conditions has become a matter of deep disappoint-

ment and has led to a slowdown in some research in

this area. Given the great complexity described above

in interpreting the mechanisms of changes in mito-

chondrial volume and related metabolism, a number

of assumptions have arisen about the active transport

of water into the mitochondrial matrix, supported by

certain contractile proteins located inside or attached

to the outside of the mitochondria [85, 86]. For exam-

ple, Lehninger, studying the mechanisms of mitochon-

drial swelling and contraction, revealed in mitochon-

dria an undialyzable thermolabile factor (contractile

factor, C-factor, reviewed in [57]), which is released

from mitochondria incubated in a medium with re-

duced glutathione (it is also released from mitochon-

dria treated by ultrasound). The addition of this factor

to the suspension of swollen mitochondria in the pres-

ence of glutathione (GSH) and ATP caused their con-

traction. However, there were strong arguments in fa-

vor of excluding such a possibility, based on the fact

that the energy required to pump water from the ma-

trix would require much more than can be obtained

during metabolism [87]. Alternative mechanisms have

been proposed, in particular, combining the immobi-

lization of solutes in the matrix and the participation

of internal hydrostatic pressure to offset the influence

of osmotic pressure [87]. Other potential elements in-

volved in violating the osmotic laws of water redistri-

bution in the matrix were called structural elements

of the matrix, which make it possible to organize a

sufficiently rigid intramitochondrial skeleton that does

not allow abrupt volume changes without their de-

struction. This is confirmed by many examples of local

rather than global matrix swelling (which again sup-

ports the claim that the mitochondria is not an ideal

osmometer) [88], as well as early data on the presence

of some elements of the intramitochondrial skeleton

obtained in electron microscopic experiments [89].

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The above rather lengthy discussion of the movement

of water between the mitochondrial matrix and the cy-

tosol does not answer the question: which route does

water use to enter and leave the mitochondria, and if

there are many of them, which of the routes provide

the main movement of water.

A new type of mitochondrial bioenergetics has

recently been described, as a result of which, due to

abundance of potassium ions over hydrogen ions in

the cytosol, ATP synthesis by mitochondrial ATP syn-

thase can be achieved by transferring potassium ions

in the ATP synthase complex from the cytosol to the

matrix [90, 91]. Theoretically, the transfer of osmotical-

ly active potassium ion associated with the operation

of the ATP synthase complex can also be associated

with the transfer of water into the mitochondrial ma-

trix. It is assumed that in this way water molecules can

be transported into the matrix, making a certain con-

tribution to the total water content in the mitochon-

drial matrix. There are at least two circumstances that

confirm this postulate. First, X-ray diffraction analysis

of the ATP synthase complex revealed water molecules

in the c-subunit of ATP synthase, which can participate

in the transport of both protons and potassium ions

[92, 93], and, secondly, general mechanisms of coupled

transport of ions and water through small channels

were described [94-98]. The assessment of the effect of

each component capable of transferring water in and

out of the mitochondria will be directly related to the

identification of a mechanism for adaptation to the en-

ergy loads of the biological system in order to ensure

a balance between energy supply and energy demand.

Generation of reactive oxygen species. It can be

agreed that one of Skulachev’s strongest achievements

was the discovery of the so-called “Skulachev ions”

together with E. A. Liberman [14], however, he himself

was a vivid apologist for the role of ROS in the vital ac-

tivity of biological systems and put this part of science

in the first place for himself.

The first work on this topic was published by Sku-

lachev in 1995 in the Russian journal Molecular Biolo-

gy [99], and it was assumed that non-phosphorylating

oxidation enables a low probability of ROS formation

in mitochondria. The idea was outwardly very simple–

when an electron moves along the respiratory chain

(or any other chain, even not associated with the final

consumption of oxygen by cytochrome oxidase), the

faster the electron runs, the less likely it is that in a

halfway to the final consumer this electron will reach

a molecule of free oxygen, eventually forming a super-

oxide anion. It is clear that in bottlenecks, where there

are actually sufficiently high levels of stationary re-

duced intermediate components of the electron trans-

port chain, this probability increases sharply, which

implies a recommendation to minimize the presence

of “bottlenecks” in order to prevent unwanted leakage

of electrons to oxygen. These bottlenecks in the ter-

minology of one of the founding fathers of the world

bioenergetics, Britton Chance, were called “cross-over

points” [100] and if within the neighboring compo-

nents of the transfer chain one is reduced and the oth-

er is oxidized, this means that communication between

these components is difficult, and this pair of carriers

reflects the limiting step in the electron transfer chain.

As a result, at these cross-over points three ADP phos-

phorylation control loci were localized, determining

the mechanism of respiratory control. In its absence

(in the case of uncoupling of oxidative phosphoryla-

tion) from the ATP synthesis, bottlenecks in the respi-

ratory chain disappear, and the generation of ROS in

these bottlenecks becomes minimal.

Later, this idea was generalized, and it became

the main guide for Skulachev’s works, advocating the

need to combat excessive generation of ROS [101].

It should be noted that at the very beginning it was

quite extreme and generally interpreted as pathogenic

any generation of ROS except that which participates

in the anti-pathogen protection organized by NADPH

oxidase which participates in phagocytosis [102]. Mito-

chondria have been declared the “dirtiest place in the

cell” to be cleared of ROS that cause oxidative changes,

including age-related damage [103]. Subsequently, the

offensive tone was softened based on the understand-

ing that ROS, in addition to the pathogenic role, also

play an important signaling role [104].

The very first fundamental works that presented

the meaning and danger of ROS generation in a living

system were published in the 40-50s of the last cen-

tury [105, 106]. Even then, the principles of the work

of antioxidants were formulated with a proposal for

their practical use to protect against oxidative damage.

In1956, Harman hypothesized the predominant role of

ROS in the aging process, which sounds as a free radi-

cal (sometimes incorrectly called mitochondrial) theory

of aging [107]. This theory has become an integral part

of a more global network theory of aging, according to

which aging is indirectly controlled by a network of

cellular and molecular defense mechanisms, including

various anti-stress reactions [108] with the later devel-

opment and isolation of the inflammatory theory of

aging [109-113].

The free radical and inflammatory theories of ag-

ing overlap on the basis of the role of mitochondria

in aging. In the first mitochondria are considered the

main place of organization of oxidative stress, and

according to the second, mitochondria are the key

place of organization of the inflammatory principle

[114,115].

Self-regulation of ROS production and energy

metabolism. Mitochondria have been declared gener-

ators and internal regulators of ROS levels in the cell

[116]. As for the generation of ROS, it is often consid-

ZOROV et al.230

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

ered that mitochondria are the main generator of ROS

in the cell. However, this is not the case, and although

mitochondria are a powerful producer of ROS, they

are not their main generators. The misconception that

mitochondria are the main site of ROS generation was

refuted in the Britton Chance group [117] with a full

description of the contribution of all cellular compo-

nents to ROS generation, among which peroxisomes

occupy the top line of the list.

It should be noted that the most cited work of

V. P. Skulachev was the establishment of a nonlinear

relationship between the magnitude of the membrane

potential and the generation of ROS in mitochondria

[118]. This led Skulachev to come to the conclusion

that it is necessary to mildly uncouple oxidative phos-

phorylation in order to prevent hyperpolarization of

mitochondria and avoid undesirable hyperproduction

of ROS [15, 39, 40, 119-121].

Detoxification. Usually, the detoxification func-

tion realized in mitochondria is limited to the synthe-

sis of urea, as a process of eliminating the decomposi-

tion products of nitrogen-containing compounds from

the biological system. The two initial stages of the urea

cycle (also called the ornithine cycle) take place in the

mitochondrial matrix and the cycle ends with reac-

tions occurring in the cytoplasm. The main source of

nitrogen bases is the product of deamination, ammo-

nia, which is eventually converted into urea, excreted

from the organism by the kidneys and which largely

serves as an indicator of the degree of normal renal

functioning. However, the detoxification process can

be considered more broadly, including biologically ac-

tive substances in the list of removal of undesirable

agents, which must perform their function in a certain

range of low concentrations, preventing their excess.

Any excess of the concentration of active substances

can be fraught with situations leading to the occur-

rence of pathologies. As a simple example, we can

consider molecules of extracellular glutamate, which

plays the role of a neurotransmitter in the brain, but

only in acceptably low concentrations. Exceeding

these concentrations outside cells is toxic (exitotoxic)

for neurons, leading to their death with a character-

istic association with nonspecific permeability of mi-

tochondria [122]. It should be noted that the level of

glutamate is largely regulated by mitochondria, name-

ly their ketoglutarate dehydrogenase [123], that is, glu-

tamate production occurs inside cells.

By the way, the detoxification principle can be ap-

plied to the above-mentioned regulation by mitochon-

dria of the level of fatty acids in the cell, which on the

one hand can be uncouplers of oxidative phosphory-

lation, and on the other, oxidation substrates.

There is a point of view that mitochondria arose

with the appearance of oxygen on earth, which has a

rather significant and poorly regulated oxidizing abil-

ity, as a result of which cellular components could be

oxidized, which is undesirable. Logically thinking, to

limit such an undesirable process, it is enough to low-

er the intracellular oxygen concentration, and mito-

chondria could perform this function. Thus, mitochon-

drial oxidative activity can be considered as a special

case of the detoxification process, and in relation to

oxygen, mitochondria can be considered as fine reg-

ulators of its concentration in accordance with the

oxygen affinity of mitochondrial cytochrome oxidase.

Thesame can be attributed to ROS, the level of which

in the cell is regulated by mitochondrial activity, subtly

balancing the necessary production of ROS and their

elimination, in particular, due to catalase [124], mito-

chondrial superoxide dismutase [125] or peroxidases

[126-128]. In addition to enzymatic systems that regu-

late the levels of intramitochondrial and intracellular

ROS, there is a whole set of low-molecular compounds

in the cell that quench the high oxidative capacity of

ROS, which can also be attributed to the detoxifica-

tion system. This part will be briefly discussed in the

next section concerning the redox buffer in the cellu-

lar system.

Creation of an intracellular redox buffer. Ho-

meostasis of the redox potential in a cell is one of the

bases of its healthy existence, and when it is disrupt-

ed, leading to temporary or chronic oxidative or re-

generative stress, several pathological situations arise.

It is known that almost all vascular pathologies of the

heart, brain and kidneys associated with ischemic ef-

fects are the result of the fact that the cellular reserves

of the redox buffer cannot cope with the oxidative

challenge, which leads to oxidation of the vital com-

ponents of the cell, requiring either internal repair or

external intervention [15, 129-131].

As we discussed above, large capacities are con-

centrated in the cell and in the mitochondria to de-

stroy excessive levels of ROS in the cell. They include,

firstly, enzymes (superoxide dismutases, catalase, per-

oxidases, ferredoxin, etc.), which are designed to neu-

tralize ROS, although not allowing an equally danger-

ous situation of ROS deficiency, which are an essential

component of cellular metabolism. Secondly, these are

small molecules united by the term antioxidants, the

chemistry of which ensures the quenching of the high

oxidative capacity of ROS. A quantitative assessment

was given in the literature [132], but it concerned the

gross antioxidant activity without specific highlighting

the contribution of partial components.

Probably, the largest contribution to antioxidant

activity is made by the proteins themselves, which car-

ry groups capable of oxidation, for example, their sulf-

hydryle groups, capable of forming an S-S transition

during oxidation. Of course, this process is highly un-

desirable in vital enzymes, given that such transitions

will inevitably affect enzymatic activity. Apparently,

MITOCENTRICITY 231

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

the evolutionary solution was to create proteins that

do not carry obvious catalytic functions, but at the ex-

pense of mass acceptance of the oxidative threat. Obvi-

ously, this is the main function of mass proteins, such

as albumins or structural proteins. Protein derivatives

and peptides play a similar role. Admittedly, reduced

GSH is the most important representative of such pep-

tides, turning into the GSSG dimer upon oxidation.

Although glutathione synthesis occurs in the cytoplasm,

in the cell it is mainly contained in the mitochondria,

where it is transported, creating the basis of the mito-

chondrial and cellular redox buffer.

In addition to glutathione, cytochrome c, localized

in the intermembrane space, can play an antioxida-

tive role in mitochondria, being associated with cyto-

chrome oxidase [133] and mitochondrial contact sites

[134, 135]. Besides the revealed peroxidase function of

cytochrome c [136] due to its high abundance in the in-

termembrane space, the massive release of this protein

from mitochondria during permeabilization of the out-

er membrane provides an intracellular concentration

sufficient to consider its contribution to direct ROS

quenching in the cell significant [137, 138]. Recently,

we hypothesized that extended mitochondrial systems

in the cell provide a more or less uniform distribution

of redox potential in the cell [139]. It should be not-

ed that for a number of years, the focus of V. P. Sku-

lachev was in the development of his own concept of

the functioning of extended mitochondrial systems as

intracellular electrical cables [140-143].

Concluding this part, which describes the main

vital functions of mitochondria, one can see how mul-

tidimensional is that part of the functioning of mito-

chondria that ensures the healthy existence of the

cell thus leading to the conclusion that homeostasis of

these vital functions is necessary. We did not review

the participation of mitochondria in the processes of

cell proliferation, differentiation, and thermogenesis,

as well as avoided examining the important role of the

mitochondrial genome, considering that these aspects

were widely considered by other researchers, but

were not included in the research scope of Skulachev

and his closest colleagues.

MITOCHONDRIA

AT THE CENTER OF DEATH

The death of a biological system implies the mul-

tilevel nature of such a specification, affecting the de-

struction of biological macromolecules (microphagy),

cellular structures (macrophagy), cells (all types of cell

death [144]), and organisms (phenoptosis). Each mech-

anism in these divisions requires an extended anal-

ysis, which, firstly, is not desirable within the frame-

work of this brief review, and, secondly, given the

focus of this work on the range of interests and works

of V. P. Skulachev, we will limit ourselves to consider-

ing the participation of mitochondria in cell death and

programmed death of the organism.

Mitochondrial and cellular death. By its name

(translated from Greek µίτος– thread and χονδρίον–

grain), mitochondria have long been considered as

very labile structures existing in the form of extended

filaments and small rounded structures, and this pro-

cess naturally raised questions – why is there a con-

stant generation of small structures that split off from

the mother tree?

Probably, now an explanation can already be found

for this. With a high probability, a constantly function-

ing electron transfer chain in mitochondria can be ac-

companied by electron leakage to various non-target

components and lead to undesirable oxidation of these

components. These oxidative damages may be repaired

or not, and in the case of the latter, a scenario for the re-

moval of these damaged components starts to emerge.

In addition to oxidized components, improperly folded

proteins and other biological structures modified by

non-oxidative origin become unwanted for mitochon-

drion, which, by an unclear mechanism, begin to seg-

regate within the same mitochondrion [145, 146]. This

process of intramitochondrial separation of “correct”

and “incorrect” elements is completed by their divi-

sion by a membrane (septum), followed by separation

of the damaged fragment and its disposal in the ma-

chinery of mitophagy. Under normal conditions, the

number of detached small fragments is not too large,

but under conditions of oxidative challenge, the en-

tire mitochondriome undergoes the process of a total

fission into fragments [147, 148], among which there

may be preserved only a part that cannot be destroyed

and which, if the oxidative challenge is eliminated,

begins to serve as the basis for building a new mito-

chondriome due to fusion with other, uncritically dam-

aged fragments. The described picture is somewhat

speculative, but it has enough arguments supported

by various data. In this whole scheme, the elimination

(death) of the mitochondria, which is called “mitopto-

sis”, is mandatory for a healthy cell. A very important

requirement for the initiation of mitochondrial death

is oxidative stress, which, as it was found, can accom-

pany the process of induction of nonspecific permea-

bility in mitochondria [149-158]. This phenomenon, at

the very beginning incomprehensible in its principle,

was characterized by the induction of a megachan-

nel in the inner mitochondrial membrane, which not

only makes it impossible for the membrane poten-

tial and ion gradients to exist, but also leads to the

high-amplitude swelling of mitochondria described

above. The latter is accompanied by permeabiliza-

tion of the outer mitochondrial membrane either due

to its rupture caused by swelling [159] and/or due to

ZOROV et al.232

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

the organization of pores formed by heteromerization

of the Bax protein [160]. Later, it has been suggested

that the induction of nonspecific permeability of mito-

chondria is a step of programmed destruction of these

organelles [161], and even later, this phenomenon was

attributed to the point of no return, preceding pro-

grammed cell death, in particular, developing by the

mechanism of necrosis [162]. The mitochondria are

critical elements that determine their own fate and the

fate of the cell, and the generally recognized concept of

mitochondrial function as the determinant of the point

of no return, that is, responsible for deciding whether

or not to be a cellular system, puts the mitochondria

at the center of a deadly cascade. To start this cascade,

the mitochondria release a number of molecules (cy-

tochrome c, AIF, procaspase IX, etc.), which alone, be-

ing inside the mitochondria, are not dangerous, but

after interaction with the components of the cytosol,

a complex is formed [163], which becomes a death

sentence for the cell. This means that mitochondria

formally carry reservoirs of cellular poisons, which

inevitably cause cell death when appropriate signals

enter the mitochondrion. Given the huge array of ex-

cellent reviews on this topic, we limit our discussion

to the above.

Death of the organism (phenoptosis). In Biochem-

istry (Moscow) special issues devoted to the phenome-

non of phenoptosis and in other publications, we have

repeatedly written about the key role of mitochondria

in this process [164-167]. The problem of phenopto-

sis, as a programmable mechanism for the death of

a multicellular organism, for couple of last decades

has been in the focus of Vladimir Skulachev, who in-

troduced mitochondria and their ROS generation into

the focus of this problem. There are several examples

of phenoptic death and they can be found in the works

of Skulachev [168-171]. It is assumed that phenoptosis

was developed during evolution to reject unneces-

sary organisms, whether sick or aged, that is, all those

that cannot compete with healthy and young organ-

isms. There are two types of phenoptosis. One caused

by stress, acute or rapid phenoptosis is a rapid dete-

rioration of the organism’s condition, subject to acute

stress. Another type, caused by age, mild or slow phe-

noptosis, is characterized by a slow deterioration of

the state, ending in the death of the organism due to

the presence of chronic stress. It follows from this that

aging and diseases associated with aging are hallmarks

of phenoptosis. Skulachev himself believed that one of

the best proofs of the involvement of mitochondria in

the programmed death of the organism were exper-

iments on the model of acute phenoptosis, in which

animals having received an almost fatal sentence as

a result of ischemia of the animal’s only kidney, sur-

vived after the introduction of mitochondrial-direct-

ed cationic agents [172]. The fact that not all of these

substances belonging to this group eliminated renal

failure, from which a fatal outcome seemed to be ex-

pected, but they all saved from death, gave a strong

evidence of the complex organization of the initiation

of a deadly cascade, possibly remote from the target

organ. This again indicated that it is the mitochondria

that determine the general poisoning of the organism

and the salvation of the latter is hidden in mitochon-

dria. Ofcourse, there were other experimental proofs

of the accuracy of the participation of mitochondria in

the death of the organism [173].

August Weismann is considered to be the founder

of the theory of programmed death [174]. However,

there is a point of view that the main ideas of pro-

grammed death of individuals were expressed earli-

er by Alfred Russel Wallace in his work Contributions

to the Theory of Natural Selection, published in 1870,

that was long before the discovery of mitochondria.

We can safely go back and assume that the ideas that

death is programmed were expressed even by the

great Russian poet Alexander S. Pushkin, who wrote

in1828:

A random and a wasted gift

Is given life, I wonder why

By some and enigmatic shift

It always is condemned to die

(translation by D. B. Zorov)

The great poet, who at the time of writing these

lines was not even 30 years old, wondered why life

was condemned to death, that is, why it was inevita-

ble. The great Russian scientist Vladimir Skulachev

came to understand the high degree of organization of

the deadly process. It allowed him to guess the basis of

this mysterious process, which he wanted to stop, can-

cel and thus to prohibit the program of death of the

organism, using knowledge of the role of mitochondria

in the organization of vital and deadly processes.

Contributions. P.A.A., N.V.A., V.A.B., L.D.Z., S.D.Z.,

I.B.P., V.A.P., D.S.S., E.I.Y., D.N.S., E.Y.P., G.T.S., and D.B.Z.

general discussion of the concept, ideology and plans

for the construction of the work; D.B.Z. writing the

manuscript; L.D.Z. and S.D.Z. editing and technical de-

sign of the manuscript.

Funding. This work was supported by the Minis-

try of Health of the Russian Federation, State Assign-

ment no.124013000594-1.

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

The authors of this work declare that they have no

conflicts of interest.

MITOCENTRICITY 233

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

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