Recent structural and biophysical studies of O2-sensing FixL, NO-sensing soluble guanylate cyclase, and other biological heme-based sensing proteins have begun to reveal the details of their molecular mechanisms and shed light on how nature regulates important biological processes such as nitrogen fixation, blood pressure, neurotransmission, photosynthesis and circadian rhythm. The O2-sensing FixL protein from S. meliloti, the eukaryotic NO-sensing protein sGC, and the CO-sensing CooA protein from R. rubrum transmit their biological signals through gas-binding to the heme domain of these proteins, which inhibits or activates the regulatory, enzymatic domain. These proteins appear to propagate their signal by specific structural changes in the heme sensor domain initiated by the appropriate gas binding to the heme, which is then propagated through a coiled-coil linker or other domain to the regulatory, enzymatic domain that sends out the biological signal. The current understanding of the signal transduction mechanisms of O2-sensing FixL, NO-sensing sGC, CO-sensing CooA and other biological heme-based gas sensing proteins and their mechanistic themes are discussed, with recommendations for future work to further understand this rapidly growing area of biological heme-based gas sensors.

Heme proteins and their derivatives play important roles in inducing lipid oxidation to produce volatile compounds during bacon drying. This study investigated the effects of heme proteins and their derivatives (hemoglobin, myoglobin, nitrosylmyoglobin, hemin, Fe2+, and Fe3+) on lipid and volatiles profiles in the washed pig muscle (WPM) model. The results of the study indicated that the inducers primarily caused the oxidation of glycerophospholipids. Furthermore, hemoglobin and myoglobin had the most significant impact, and their potential substrates may include PE (O-18:2/20:4), PE (O-18:1/20:4), PC (16:0/18:1), and PE (O-18:2/18:2). Nitrosomyoglobin has limited ability to promote lipid oxidation and may protect ether phospholipids from oxidation. The analysis of the volatiles in the model revealed that heme proteins and their derivatives have the ability to induce the production of key aroma compounds. The descending order of effectiveness in inducing the production of aroma compounds is as follows: hemoglobin, myoglobin, hemin, and nitrosylmyoglobin. The effectiveness of Fe2+ and Fe3+ is similar to that of nitrosylmyoglobin.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease.

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme‑copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 μM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 μM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 μM CO.

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect\'s salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.

The scaly-foot gastropod (Chrysomallon squamiferum), which lives in the deep-sea zone of oceans around thermal vents, has a black shell and scales on the foot. Both the black shell and scales contain iron sulfide minerals such as greigite (Fe3S4) and pyrite (FeS2). Although pyrite nanoparticles can be used as materials for solar panels, it is difficult to synthesize stable and spherical nanoparticles in vitro. In this study, we extracted organic molecules that interact with nano-pyrite from the shell of the scaly-foot gastropod to develop a low-cost, eco-friendly method for pyrite nanoparticles synthesis. Myoglobin (csMG), a heme protein, was identified in the iron sulfide layer of the shell. We purified recombinant csMG (r-csMG) and demonstrated that r-csMG helped in the conversion of ferric ions, sulfide ions and sulfur into spherical shaped pyrite nanoparticles at 80°C. To reduce the effort and cost of production, we showed that commercially available myoglobin from Equus caballus (ecMG) also induced the in vitro synthesis of pyrite nanoparticles. Using structure-function experiments with digested peptides, we highlighted that the amino acid sequence of r-csMG peptides controlled the spherical shape of the nanoparticle while the hemin molecules, which the peptides interacted with, maintained the size of nanoparticles. Synthesized pyrite nanoparticles exhibited strong photoluminescence in the visible wavelength region, suggesting its potential application as a photovoltaic solar cell material. These results suggest that materials for solar cells can be produced at low cost and energy under eco-friendly conditions. STATEMENT OF SIGNIFICANCE: Pyrite is a highly promising material for photovoltaic devices because of its excellent optical, electrical, magnetic, and transport properties and high optical absorption coefficient. Almost all current pyrite synthesis methods use organic solvents at high temperature and pressure under reducing conditions. Synthesized pyrite nanoparticles are unstable and are difficult to use in devices. The scaly-foot gastropod can synthesize pyrite nanoparticles in vivo, meaning that pyrite nanoparticles can be generated in an aqueous environment at low temperature. In this study, we demonstrated the synthesis of pyrite nanoparticles using a heme protein identified in the iron sulfide layer of the scaly-foot gastropod shell. These results exemplify how natural products in organisms can inspire the innovation of new technology.