Investigating Cytoglobin Expression in Colon Cancer: Clinicopathological Insights from Immunohistochemical Analysis.

BACKGROUND/AIM: Cytoglobin (Cygb), a protein involved in cellular oxygen metabolism and protection, has garnered attention owing to its potential role in the initiation and progression of cancer, particularly colon cancer (CC). This study investigated the expression and significance of Cygb in CC. PATIENTS AND METHODS: This study included 145 patients who underwent R0 surgery for CC (clinical stage II/III) at our institution between January 2007 and December 2014. Immunohistochemical analysis was performed to evaluate the Cygb expression patterns in CC tissues. Additionally, the correlation between Cygb expression levels and the clinicopathological characteristics of patients with CC was investigated. RESULTS: Colon cancer tissues were categorized into high-expression (95 cases) and low-expression (50 cases) groups. Cygb was highly expressed in well-differentiated cases, whereas its expression decreased in poorly differentiated cases. No significant differences in other clinicopathological factors were observed between the two groups. Cygb expression had no significant effect on recurrence-free survival or overall survival. CONCLUSION: This study contributes to the growing understanding of Cygb expression and its significance in CC. The expression of Cygb in CC was found to be unrelated to the recurrence rate and prognosis, but showed a correlation with differentiation status.

Transcriptomics of a cytoglobin knockout mouse: Insights from hepatic stellate cells and brain.

The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.

The knockout of cytoglobin 1 in zebrafish (Danio rerio) alters lipid metabolism, iron homeostasis and oxidative stress response.

Cytoglobin (Cygb) is an evolutionary ancient heme protein with yet unclear physiological function(s). Mammalian Cygb is ubiquitously expressed in all tissues and is proposed to be involved in reactive oxygen species (ROS) detoxification, nitric oxide (NO) metabolism and lipid-based signaling processes. Loss-of-function studies in mouse associate Cygb with apoptosis, inflammation, fibrosis, cardiovascular dysfunction or oncogenesis. In zebrafish (Danio rerio), two cygb genes exist, cytoglobin 1 (cygb1) and cytoglobin 2 (cygb2). Both have different coordination states and distinct expression sites within zebrafish tissues. The biological roles of the cygb paralogs are largely uncharacterized. We used a CRISPR/Cas9 genome editing approach and generated a knockout of the penta-coordinated cygb1 for in vivo analysis. Adult male cygb1 knockouts develop phenotypic abnormalities, including weight loss. To identify the molecular mechanisms underlying the occurrence of these phenotypes and differentiate between function and effect of the knockout we compared the transcriptomes of cygb1 knockout at different ages to age-matched wild-type zebrafish. We found that immune regulatory and cell cycle regulatory transcripts (e.g. tp53) were up-regulated in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defense and iron homeostasis was affected in the cygb1 knockout. Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and may be involved in lipid-, iron-, and ROS-dependent signaling.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Effect of Globin Digests on Physical Fatigue in Mice.

Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.

CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation.

Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.

A novel single sensor hemoglobin domain from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 exhibits higher pH but lower thermal stability compared to globins from mesophilic organisms.

Thermosynechococcus elongatus-BP1 belongs to the class of photoautotrophic cyanobacterial organisms. The presence of chlorophyll a, carotenoids, and phycocyanobilin are the characteristics that categorize T. elongatus as a photosynthetic organism. Here, we report the structural and spectroscopic characteristics of a novel hemoglobin (Hb) Synel Hb from T.elongatus, synonymous with Thermosynechococcus vestitus BP-1. The X-ray crystal structure (2.15 Å) of Synel Hb suggests the presence of a globin domain with a pre-A helix similar to the sensor domain (S) family of Hbs. The rich hydrophobic core accommodates heme in a penta-coordinated state and readily binds an extraneous ligand (imidazole). The absorption and circular dichroic spectral analysis of Synel Hb reiterated that the heme is in FeIII+ state with a predominantly α-helical structure similar to myoglobin. Synel Hb displays higher resistance to structural perturbations induced via external stresses like pH and guanidium hydrochloride, which is comparable to Synechocystis Hb. However, Synel Hb exhibited lower thermal stability compared to mesophilic hemoglobins. Overall, the data is suggestive of the structural sturdiness of Synel Hb, which probably corroborates its origin in extreme thermophilic conditions. The stable globin provides scope for further investigation and may lead to new insights with possibilities for engineering stability in hemoglobin-based oxygen carriers.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3-. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2- to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M-1 s-1, 9.3 M-1 s-1, 1.4 × 101 M-1 s-1, and 5.8 M-1 s-1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2--based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-ß-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-ß-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.

A Novel Resveratrol-Induced Pathway Increases Neuron-Derived Cell Resilience against Oxidative Stress.

A promising therapeutic strategy to delay and/or prevent the onset of neurodegenerative diseases (NDs) could be to restore neuroprotective pathways physiologically triggered by neurons against stress injury. Recently, we identified the accumulation of neuroglobin (NGB) in neuronal cells, induced by the 17ß-estradiol (E2)/estrogen receptor ß (ERß) axis, as a protective response that increases mitochondria functionality and prevents the activation of apoptosis, increasing neuron resilience against oxidative stress. Here, we would verify if resveratrol (Res), an ERß ligand, could reactivate NGB accumulation and its protective effects against oxidative stress in neuronal-derived cells (i.e., SH-SY5Y cells). Our results demonstrate that ERß/NGB is a novel pathway triggered by low Res concentrations that lead to rapid and persistent NGB accumulation in the cytosol and in mitochondria, where the protein contributes to reducing the apoptotic death induced by hydrogen peroxide (H2O2). Intriguingly, Res conjugation with gold nanoparticles increases the stilbene efficacy in enhancing neuron resilience against oxidative stress. As a whole, ERß/NGB axis regulation is a novel mechanism triggered by low concentration of Res to regulate, specifically, the neuronal cell resilience against oxidative stress reducing the triggering of the apoptotic cascade.

Neuroglobin Facilitates Neuronal Oxygenation through Tropic Migration under Hypoxia or Anemia in Rat: How Does the Brain Breathe?

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His64) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.

Resveratrol Analogs and Prodrugs Differently Affect the Survival of Breast Cancer Cells Impairing Estrogen/Estrogen Receptor α/Neuroglobin Pathway.

Breast cancer is the first leading tumor in women in terms of incidence worldwide. Seventy percent of cases are estrogen receptor (ER) α-positive. In these malignancies, 17ß-estradiol (E2) via ERα increases the levels of neuroglobin (NGB), a compensatory protein that protects cancer cells from stress-induced apoptosis, including chemotherapeutic drug treatment. Our previous data indicate that resveratrol (RSV), a plant-derived polyphenol, prevents E2/ERα-induced NGB accumulation in this cellular context, making E2-dependent breast cancer cells more prone to apoptosis. Unfortunately, RSV is readily metabolized, thus preventing its effectiveness. Here, four different RSV analogs have been developed, and their effect on the ERα/NGB pathway has been compared with RSV conjugated with highly hydrophilic gold nanoparticles as prodrug to evaluate if RSV derivatives maintain the breast cancer cells' susceptibility to the chemotherapeutic drug paclitaxel as the original compound. Results demonstrate that RSV conjugation with gold nanoparticles increases RSV efficacy, with respect to RSV analogues, reducing NGB levels and enhancing the pro-apoptotic action of paclitaxel, even preventing the anti-apoptotic action exerted by E2 treatment on these cells. Overall, RSV conjugation with gold nanoparticles makes this complex a promising agent for medical application in breast cancer treatment.

Interaction of nitrite with ferric protoglobin from Methanosarcina acetivorans - an interesting model for spectroscopic studies of the haem-ligand interaction.

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin belonging to the same lineage of the globin superfamily as globin-coupled sensors. A putative role in the scavenging of reactive nitrogen and oxygen species has been suggested as a possible adaptation mechanism of the host organism to different gaseous environments in the course of evolution. A combination of optical absorption, electronic circular dichroism (ECD), resonance Raman (rRaman), and electron paramagnetic resonance (EPR) reveal the unusual in vitro reaction of ferric MaPgb with nitrite. In contrast to other globins, a large excess of nitrite did not induce the formation of a nitriglobin form in MaPgb. Surprisingly, the addition of nitrite in mildly acidic pH led to the formation of a stable nitric-oxide ligated ferric form of the protein (MaPgb-NO). Furthermore, the 300-700 nm ECD spectrum of ferric MaPgb is for the first time reported and discussed, showing strong differences in the Soret and Q ellipticity compared to ferric myoglobin, in line with the unusually strongly ruffled haem group of MaPgb and the related quantum-mechanical admixture of the S = 5/2 and S = 3/2 state of its ferric form. The Soret and Q ellipticity change strongly upon formation of MaPgb-NO, revealing a significant effect of the nitric-oxide ligation on the haem group and pocket. The related changes in the asymmetric pyrrole half-ring stretching vibration modes observed in the rRaman spectra give experimental support to earlier theoretical models, in which an important role of the in-plane breathing modes of the haem was predicted for the stabilization of the binding of diatomic gases to MaPgb.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis.

Soy leghemoglobin is a key food additive that imparts meaty flavor and color to meat analogs. Here, a Pichia pastoris strain capable of high-yield secretory production of functional leghemoglobin was developed through gene dosage optimization and heme pathway consolidation. First, multi-copy integration of LegH expression cassette was achieved via both post-transformational vector amplification and CRISPR/Cas9 mediated genome editing methods. A combination of inducible expression and constitutive expression resulted in the highest production of leghemoglobin. Then, heme biosynthetic pathway was engineered to address challenges in heme depletion and leghemoglobin secretion. Finally, the disruption of ku70 was complemented in engineered P. pastoris strain to enable high-density fermentation in a 10-L bioreactor. These engineering strategies increased the secretion of leghemoglobin by more than 83-fold, whose maximal leghemoglobin titer and heme binding ratio reached as high as 3.5 g/L and 93 %, respectively. This represents the highest secretory production of heme-containing proteins ever reported.

Evolutionary analysis of globin domains from kinetoplastids.

Globin (Gb) domains function in sensing gaseous ligands like oxygen and nitric oxide. In recent years, Gb domain containing heme binding adenylate cyclases (OsAC or GbAC) emerged as significant modulator of Leishmania response to hypoxia and oxidative stress. During progression of life cycle stages, kinetoplastids experience altered condition in insect vectors or other hosts. Moreover, marked diversity in life style has been accounted among kinetoplastids. Distribution and abundance of Gb-domains vary between different groups of kinetoplastids. While in bodonoids, Gbs are not combined with any other functional domains, in trypanosomatids it is either fused with adenylate cyclase (AC) or oxidoreductase (OxR) domains. In salivarian trypanosomatids and Leishmania (Viannia) subtypes, no gene product featuring Gbs can be identified. In this context, evolution of Gb-domains in kinetoplastids was explored. GbOxR derived Gbs clustered with bacterial flavohemoglobins (fHb) including one fHb from Advenella, an endosymbiont of monoxeneous trypanosomatids. Codon adaptation and other evolutionary analysis suggested that OsAC (LmjF.28.0090), the solitary Gb-domain featuring gene product in Leishmania, was acquired via possible horizontal gene transfer. Substantial functional divergence was estimated between orthologues of genes encoding GbAC or GbOxR; an observation also reflected in structural alignment and heme-binding residue predictions. Orthologue-paralogue and synteny analysis indicated genomic reduction in GbOxR and GbAC loci for dixeneous trypanosomatids.

Heme as a differentiation-regulatory transcriptional cofactor.

The hematopoietic transcription factor GATA1 induces heme accumulation during erythropoiesis by directly activating genes mediating heme biosynthesis. In addition to its canonical functions as a hemoglobin prosthetic group and enzyme cofactor, heme regulates gene expression in erythroid cells both transcriptionally and post-transcriptionally. Heme binding to the transcriptional repressor BACH1 triggers its proteolytic degradation. In heme-deficient cells, BACH1 accumulates and represses transcription of target genes, including α- and ß-like globin genes, preventing the accumulation of cytotoxic free globin chains. A recently described BACH1-independent mechanism of heme-dependent transcriptional regulation is associated with a DNA motif termed heme-regulated motif (HERM), which resides at the majority of loci harboring heme-regulated chromatin accessibility sites. Progress on these problems has led to a paradigm in which cell type-specific transcriptional mechanisms determine the expression of enzymes mediating the synthesis of small molecules, which generate feedback loops, converging upon the transcription factor itself and the genome. This marriage between transcription factors and the small molecules that they control is predicted to be a canonical attribute of regulatory networks governing cell state transitions such as differentiation in the hematopoietic system and more broadly.

Neuroglobin Is Involved in the Hypoxic Stress Response in the Brain.

Neuroglobin is an oxygen-binding heme protein expressed predominantly in the brain. Despite many years of research, the exact distribution and expression of neuroglobin in the neocortical development and under mild hypoxia stress still remain unclear. Therefore, we aim to explore the expression of neuroglobin during neocortex expansion and under mild hypoxia stress in vivo. We used Kunming mice to examine the expression of Ngb protein during neocortex expansion. In addition, we analyzed the density of Ngb-positive neural stem cells using the Image-Pro PLUS (v.6) computer software program (Media Cybernetics, Inc.). Our data indicated that the density of the neuroglobin-positive neurons in mice cerebral cortex displayed a downward trend after birth compared with high expression of neuroglobin in a prenatal period. Similarly, we identified that neurons were capable of ascending neuroglobin levels in response to mild hypoxic stress compared with the no intervention group. These findings suggest that neuroglobin behaves as a compensatory protein regulating oxygen provision in the process of neocortical development or under physiological hypoxia, further contributing to the discovery of novel therapeutic methods for neurological disorders, which is clinically important.

Editorial for Special Issue: Neuroglobin from Brain Protection to Cancer Progression.

Since its discovery in 2000 [...].

Androglobin, a chimeric mammalian globin, is required for male fertility.

Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.

Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations.

Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M-1s-1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions.