Flavin Mononucleotide Cofactor Research Articles

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I.

The flavin mononucleotide (FMN) cofactor of respiratory complex I occupies a key position in the electron transport chain. Here, the electrons coming from NADH start the sequence of oxidoreduction reactions, which drives the generation of the proton-motive force necessary for ATP synthesis. The overall architecture and the general catalytic proprieties of the FMN site are mostly well established. However, several aspects regarding the complex I flavin cofactor are still unknown. For example, the flavin binding to the N-module, the NADH-oxidizing portion of complex I, lacks a molecular description. The dissociation of FMN from the enzyme is beginning to emerge as an important regulatory mechanism of complex I activity and ROS production. Finally, how mitochondria import and metabolize FMN is still uncertain. This review summarizes the current knowledge on complex I flavin cofactor and discusses the open questions for future research.

Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart

Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.

Recent trends in enzyme engineering aiming to improve bioelectrocatalysis proceeding with direct electron transfer

Among the known types of electrochemical biosensors, the third generation based on the ability of some enzymes to direct electron transfer (DET) is the most promising one. The enzyme property to DET is depending on its capability to electron transfer from enzymatically reduced built-in native cofactor (flavin mononucleotide, flavin adenine dinucleotide, pyrroloquinoline quinone, or heme) to a conductive surface directly for single cofactor enzymes or through a native structural electron acceptor (heme or copper-containing prosthetic groups) for multicofactor enzymes. Thus, there are two possibilities to use such type enzymes: to find a natural source of the enzyme with these properties; or to construct the recombinant chimeric analogs using the gene-engineering techniques. The modern molecular genetics opens the possibility to be independent of million-year natural evolution and engineer the specific enzymes for scientific and technological needs. This brief review is focused mostly on the recent publications on application of DET-capable engineered enzymes for the third-generation electrochemical biosensors.

Kinetics and thermodynamics of enzymatic decarboxylation of α,β-unsaturated acid: a theoretical study.

Enzymatic decarboxylation of α,β-unsaturated acid through ferulic acid decarboxylase (FDC1) has been of interest because this reaction has been anticipated to be a promising, environmentally friendly industrial process for producing styrene and its derivatives from natural resources. Because the local dielectric constant at the active site is not exactly known, enzymatic decarboxylation to generate β-methylstyrene (β-MeSt) was studied under two extreme conditions (ε = 1 and 78 in the gas phase and aqueous solution, respectively) using the B3LYP/DZP method and transition state theory (TST). The model molecular clusters consisted of an α-methylcinnamate (Cin) substrate, a prenylated flavin mononucleotide (PrFMN) cofactor and all relevant residues of FDC1. Analysis of the equilibrium structures showed that the FDC1 backbone does not play the most important role in the decarboxylation process. The potential energy profiles confirmed that the increase in the polarity of the solvent could lead to significant changes in the energy barriers, especially for the transition states that involve proton transfer. Analysis of the rate constants confirmed the low/no quantum mechanical tunneling effect in the studied temperature range and that inclusion of the fluctuation of the local dielectric environment in the mechanistic model was essential. Because the computed rate constants are not compatible with the time resolution of the stopped-flow spectrophotometric experiment, the direct route for generating β-MeSt after CO2 elimination (acid catalyst (2)) is unlikely to be utilized, thereby confirming that indirect cycloelimination in a low local dielectric environment is the rate determining step. The thermodynamic results showed that the elementary reactions that involve charge (proton) transfer are affected by solvent polarity, thereby leading to the conclusion that overall, the enzymatic decarboxylation of α,β-unsaturated acid is thermodynamically controlled at high ε. The entropy changes due to the generation of molecules in the active site appeared more pronounced than that due to only covalent bond breaking/formation or structural reorientation. This work examined in detail for the first time the scenarios in each elementary reaction and provided insight into the effect of the fluctuations in the local dielectric environment on the enzymatic decarboxylation of α,β-unsaturated acids. These results could be used as guidelines for further theoretical and experimental studies on the same and similar systems.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase

BackgroundTuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb.ResultsIn this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways.ConclusionMSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Membrane-Bound Flavocytochrome MsrQ Is a Substrate of the Flavin Reductase Fre in Escherichia coli.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

Influence of Magnetic Fields on Electrochemical Reactions of Redox Cofactor Solutions

AbstractRedox cofactors mediate many enzymatic processes and are increasingly employed in biomedical and energy applications. Exploring the influence of external magnetic fields on redox cofactor chemistry can enhance our understanding of magnetic‐field‐sensitive biological processes and allow the application of magnetic fields to modulate redox reactions involving cofactors. Through a combination of experiments and modeling, we investigate the influence of magnetic fields on electrochemical reactions in redox cofactor solutions. By employing flavin mononucleotide (FMN) cofactor as a model system, we characterize magnetically induced changes in Faradaic currents. We find that radical pair intermediates have negligible influence on current increases in FMN solution upon application of a magnetic field. The dominant mechanism underlying the observed current increases is the magneto‐hydrodynamic effect. We extend our analyses to other diffusion‐limited electrochemical reactions of redox cofactor solutions and arrive at similar conclusions, highlighting the opportunity to use this framework in redox cofactor chemistry.

Electrostatics and water occlusion regulate covalently-bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR.

Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one-electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH2 , but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

Mechanistic Study of Oxidoreductase AprQ Involved in Biosynthesis of Aminoglycoside Antibiotic Apramycin

Main observation and conclusionThe aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety, and biosynthesis of this moiety involves an oxidoreductase AprQ. Unlike other known “Q” series proteins involved in aminoglycosides biosynthesis, AprQ does not work with an aminotransferase partner, and performs a four‐electron oxidation that converts a CH2OH moiety to a carboxylate group. In this study, we report mechanistic investigation of AprQ. We showed AprQ contains a flavin mononucleotide (FMN) cofactor, which is different from other known Q series enzymes that contain a flavin adenine dinucleotide (FAD) cofactor. A series of biochemical assays showed that AprQ is not a monooxygenase but a flavoprotein oxidase. Although molecular O2 is strictly required for reaction turnover, four‐electron oxidation can be achieved in the absence of O2 in single turnover condition. These findings establish the detailed catalytic mechanism of AprQ and expand the growing family of flavoprotein oxidases, an increasingly important class of biocatalysts.

Site-Specific Photoinduced Dynamics of the LOV Protein EL222 Monitored by Multiple Genetically Encoded Infrared Probes

Proteins containing the light-oxygen-voltage (LOV) domain are photosensory receptors that mediate biological actions in response to blue light. For instance, the bacterial transcription factor EL222 regulates gene expression in a light-regulated manner and has found use in optogenetic applications. Upon excitation of the embedded flavin mononucleotide (FMN) cofactor, EL222 undergoes structural changes that ultimately drive its association with DNA. However, our knowledge of the light-adapted state(s) and the molecular mechanism underlying the transition between dark and lit states is incomplete.

Alteration of Flavin Cofactor Homeostasis in Human Neuromuscular Pathologies.

The aim of this short review chapter is to provide a brief summary of the relevance of riboflavin (Rf or vitamin B2) and its derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for human neuromuscular bioenergetics.Therefore, as a completion of this book we would like to summarize what kind of human pathologies could derive from genetic disturbances of Rf transport, flavin cofactor synthesis and delivery to nascent apoflavoproteins, as well as by alteration of vitamin recycling during protein turnover.

Comparison of biotransformation mechanisms of 2, 4, 6-trinitrotoluene and its hydride-Meisenheimer metabolite by the old yellow enzyme family of flavoproteins

2,4,6-trinitrotoluene (TNT) is a persistent pollutant; for removing it from environment, the biodegradation becomes an economical and environmentally friendly alternative. However, TNT is difficult to naturally mineralize due to the stability of benzene ring. Interestingly, the hydride metabolite (Hˉ-TNT) of TNT can produce dearomatized products by the biotransformation of old yellow enzyme (OYE) family, which is highly different with TNT. It is a promising strategy for the ring-opening degradation of TNT. Here, we explore the biotransformation difference of TNT and Hˉ-TNT by OYE family. The results show that the electron-withdrawing ability of nitro groups makes them obtain the majority of negative charges of Hˉ-TNT, which promotes the strong interaction between the substrate and key residues. It in turn promotes the formation of π-π stacking configuration between Hˉ-TNT and the flavin mononucleotide (FMN) cofactor, which is a precondition of the aromatic ring reduction. As a result, the aromatic ring reduction occupies an absolute advantage, while the nitro reduction is suppressed. The conclusions are in line with previous understanding and can explain experimental phenomena. This work can provide a valuable reference for the combined strategy of mineralizing TNT.

Retbindin: A riboflavin Binding Protein, Is Critical for Photoreceptor Homeostasis and Survival in Models of Retinal Degeneration.

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models; one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn−/− and Prph2Y141C/+/Rtbdn−/− retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

Ferulic Acid Decarboxylase Controls Oxidative Maturation of the Prenylated Flavin Mononucleotide Cofactor.

Prenylated flavin mononucleotide (prFMN) is a recently discovered modified flavin cofactor containing an additional nonaromatic ring, connected to the N5 and C6 atoms. This cofactor underpins reversible decarboxylation catalyzed by members of the widespread UbiD enzyme family and is produced by the flavin prenyltransferase UbiX. Oxidative maturation of the UbiX product prFMNH2 to the corresponding oxidized prFMNiminium is required for ferulic acid decarboxylase (Fdc1; a UbiD-type enzyme) activity. However, it is unclear what role the Fdc1 enzyme plays in this process. Here, we demonstrate that, in the absence of Fdc1, prFMNH2 oxidation by O2 proceeds via a transient semiquinone prFMNradical species and culminates in a remarkably stable prFMN-hydroperoxide species. Neither forms of prFMN are able to support Fdc1 activity. Instead, enzyme activation using O2-mediated oxidation requires prFMNH2 binding prior to oxygen exposure, confirming that UbiD enzymes play a role in O2-mediated oxidative maturation. In marked contrast, alternative oxidants such as potassium ferricyanide support prFMNiminium formation both in solution and in Fdc1.

Ultrafast Vibrational Energy Transfer between Protein and Cofactor in a Flavoenzyme.

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond–picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable “heavy” isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive flavoenzymes.

Encapsulation of Flavin Cofactor within a Manganese Porphyrin-Based Metal-Organic Polyhedron for Reductive Dioxygen Activation.

Encapsulation of flavin mononucleotide (FMN) in a porphyrinatomanganese(III)-based cubic cage allowed the fast reduction of manganese(III) porphyrin in the presence of nicotinamide adenine dinucleotide (NADH). This supramolecular system was capable of efficiently activating dioxygen and catalyzing the oxidation of benzyl alcohol. Control experiments suggested that the close proximity between FMN and manganese(III) porphyrins forced by the host-guest interaction might benefit the electron-transfer process from the FMN cofactor to the metal centers.

A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption.

Time-resolved infrared spectroscopy on irreversible reactions requires in general an exchange of sample for thousands of acquisitions leading to high sample consumption. Here, we present a setup employing a modern quantum cascade laser (QCL) as a probe light source to record time-resolved difference spectra of irreversible photoreactions in H2O. The combination of the focused QCL with a pressure-tolerant flow cell and a micrometre stage orthogonal to the flow allowed us to drastically reduce the sample consumption. We investigated the irreversible photoreduction of the cofactor flavin mononucleotide (FMN) in H2O, which is a common reaction taking place in biological photoreceptors. A broad time range from 20 nanoseconds to 1 second was accessible, because the approach minimized any signal drift by the flow. Kinetics were recorded at 46 selected wavenumbers consuming 12 microlitres for a complete dataset. The tuning range of 1490-1740 cm-1 included relevant carbonyl vibrations and the region of strong water absorption at around 1650 cm-1. A continuous dataset in the spectral dimension was generated by applying a fit with a sum of Lorentzians. Subsequent global analysis allowed us to resolve reference spectra and kinetics of the photoreaction proceeding from the triplet excited state via the intermediate flavin anion radical to the product, the fully reduced state of FMN. Accordingly, the neutral radical state is not populated in the disproportionation. The approach strongly facilitates the spectroscopic access to irreversible reactions of flavin-containing photoreceptors and photoenzymes with high time resolution and small sample consumption.

Influence of the cofactor structure on the photophysical processes initiating signal transduction in a phototropin-derived LOV domain.

Due to their biological importance, the photochemistry of blue-light photoreceptor proteins has been studied extensively over the last few decades. Most blue-light photoreceptors, such as cryptochromes and phototropins, utilize flavin chromophores as their cofactors. After irradiation with light, the chromophore undergoes electron transfer with nearby redox-active amino-acid residues within the protein, whereby this first step of signal transduction may be initiated either from the flavin's excited singlet or triplet state. Despite the collective effort of theoreticians and experimentalists to characterize and understand the photochemistry of flavoproteins, the mechanistic details of the excited state processes initiating signal transduction are yet to be revealed. Here, we use a light-oxygen-voltage-sensing domain from Avena sativa phototropin to get additional insight into the excited state photochemistry of flavoproteins. The influence of structural variations of the cofactor flavin mononucleotide (FMN) is explored by varying the methyl substitution pattern in positions 7 and 8 of the flavin core. The photophysical properties of the FMN derivatives, in the absence and presence of the protein environment, are investigated by UV-vis absorption, fluorescence, and electron paramagnetic resonance spectroscopies as well as cyclic voltammetry. The comparison of the properties of the modified flavin cofactors with those of FMN shows that the rates of the different excited state reactions, and therefore also the singlet/triplet yields, can be modulated substantially by only minor structural modifications of the flavin core.

An exclusive computational insight toward molecular mechanism of MMV007571, a multitarget inhibitor of Plasmodium falciparum

Recently, two Malaria Box molecules namely MMV007571 and MMV020439 well known inhibitors of New Permeability Pathway (NPP) function also showed a secondary phenotype of inhibition of enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) and cytochrome bc1 complex in metabolic profile assays. Intricacies of their binding at the newly identified targets was need of the hour which motivated us to study their binding using molecular docking and dynamics simulations approach. Interestingly, molecular docking results of both MMV007571 and MMV020439 showed good binding affinity toward the Qo site of cytochrome bc1 complex while only MMV007571 illustrated notable binding characterstics for PfDHODH. Molecular Dynamics (MD) simulations when carried out for native-PfDHODH, PfDHODH-MMV007571 and PfDHODH-Genz667348 models (100 ns each) demonstrated the role of inhibitors over the N-terminus domain which experienced conformational transition from an open state (22 Å) to closed state (16 Å) in the protein-inhibitor models. Dynamics also indicated that the loop domain near cofactor flavin mononucleotide (FMN) attained more felxibility which further lead to its poor binding and may contribute to inhibition of the oxidation (catalytic) process. Moreover, the pharmacophoric features of MMV007571 was justified and may serve as a template for the design of novel series of more potent multitarget inhibitors against Plasmodium falciparum.AbbreviationsÅAngstromACTsArtemisinin combination therapiescyt bc1cytochrome bc1 complexhhour(s)KKelvinµMmicromolarMMVMedicine for malaria ventureNLucNanoluciferasenMnanomolarNPPNew permeation pathwayPDBProtein data bankPfDHODHPlasmodium falciparum dihydroorotate dehydrogenasePOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholineRBCRed blood corpusclesRMSDRoot-mean-square deviationSPStandard precisionvdWvan der WaalsXPExtra precisionyDHODHYeast dihydroorotate dehydrogenaseCommunicated by Ramaswamy H. Sarma

Brain Ischemia/Reperfusion Injury and Mitochondrial Complex I Damage.

Ischemic stroke and neonatal hypoxic-ischemic encephalopathy are two of the leading causes of disability in adults and infants. The energy demands of the brain are provided by mitochondrial oxidative phosphorylation. Ischemia/reperfusion (I/R) affects the production of ATP in brain mitochondria, leading to energy failure and death of the affected tissue. Among the enzymes of the mitochondrial respiratory chain, mitochondrial complex I is the most sensitive to I/R; however, the mechanisms of its inhibition are poorly understood. This article reviews some of the existing data on the mitochondria impairment during I/R and proposes two distinct mechanisms of complex I damage emerging from recent studies. One mechanism is a reversible dissociation of natural flavin mononucleotide cofactor from the enzyme I after ischemia. Another mechanism is a modification of critical cysteine residue of complex I involved into the active/deactive conformational transition of the enzyme. I describe potential effects of these two processes in the development of mitochondrial I/R injury and briefly discuss possible neuroprotective strategies to ameliorate I/R brain injury.