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Magnesium (Mg 2+ ) plays a critical role in many physiological processes. The AtMRS2/MGT family, which contains nine Arabidopsis genes (and two pseudogenes), belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-11/MGT10 possesses the signature GlyMetAsn sequence (the GMN motif) conserved in the CorA superfamily; however, little is known about the role of the GMN motif in AtMRS2. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-11 mediated rapid Mg 2+ uptake into proteoliposomes at extraliposomal Mg 2+ concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg 2+ uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg 2+ uptake. The functional complementation assay in Escherichia coli strain TM2 showed that E. coli cells expressing AtMRS2-11 with mutations in G of the GMN motif did not grow in LB medium without Mg 2+ supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg 2+ ; no difference was observed between the growth of TM2 cells expressing the AtMRS2-11 G417W mutant and that of cells expressing wild-type AtMRS2-11. These results suggested that the Mg 2+ transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg 2+ concentrations; thus, the Gly residue is critical for Mg 2+ transport, and the Mg 2+ transport activity of the GMN-motif mutants was increased at high Mg 2+ concentrations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca 2+ -dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity ( Kd = 32.4 nM) in the presence of Ca 2+ . The heat capacity change (Δ Cp ) of the reaction showed a large negative value (-390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.

Magnesium (Mg 2+ ) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg 2+ uptake into proteoliposomes. The rapid Mg 2+ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni 2+ transport activity but almost no Co 2+ transport activity. The rapid Mg 2+ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg 2+ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg 2+ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Circadian rhythms are regulated by transcription-translation feedback loops (TTFL) of clock genes. Previous studies have demonstrated that core transcriptional factors, NPAS2 and CLOCK, in the TTFL can reversibly bind carbon monoxide (CO) in vitro. However, little is known about whether endogenous CO, which is continuously produced during a heme metabolic process, is involved in the circadian system. Here we show that selective removal of endogenous CO in mice considerably disrupts rhythmic expression of the clock genes. A highly selective CO scavenger, hemoCD1, which is a supramolecular complex of an iron(II)porphyrin with a per-O-methyl-β-cyclodextrin dimer, was used to remove endogenous CO in mice. Intraperitoneal administration of hemoCD1 to mice immediately reduced the amount of internal CO. The removal of CO promoted the bindings of NPAS2 and CLOCK to DNA (E-box) in the murine liver, resulting in up-regulation of the E-box-controlled clock genes (Per1, Per2, Cry1, Cry2, and Rev-erbα). Within 3 h after the administration, most hemoCD1 in mice was excreted in the urine, and heme oxygenase-1 (HO-1) was gradually induced in the liver. Increased endogenous CO production due to the overexpression of HO-1 caused dissociation of NPAS2 and CLOCK from E-box, which in turn induced down-regulation of the clock genes. The down-regulation continued over 12 h even after the internal CO level recovered to normal. The late down-regulation was ascribed to an inflammatory response caused by the endogenous CO reduction. The CO pseudo-knockdown experiments provided the clear evidence that endogenous CO contributes to regulation in the mammalian circadian clock.

Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Magnesium (Mg2+) plays a critical role in many physiological processes. Mg2+ transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg2+ transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10 mM Mg2+ to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg2+ concentration. The Mg2+ requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg2+ transport under high Mg2+ growth conditions in E. coli. The Arabidopsis Mg2+ transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1 mM Mg2+ and without Mg2+ supplementation, respectively, and cell growth was inhibited by 2 mM AlCl3. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg2+ and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Several factors involved in the core circadian rhythm are PAS domain proteins, one of which, neuronal PAS2 (NPAS2), contains a heme-binding motif. It is thought that heme controls the transcriptional activity of core circadian factors BMAL1-NPAS2, and that the heme-binding nuclear receptor REV-erbα negatively regulates the expression of BMAL1. To examine the role of heme in the nucleus, we expressed nuclear hemeproteins including the nuclear localization signal-added cytoglobin, NPAS2 and REV-erbα. Then, the living cells expressing these proteins were treated with 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). The fluorescent signal derived from DCFH-DA was observed in the nucleus. When the cells were cultured with hemin, the signal of heme in the nucleus increased. Considering that DCFH-DA reacted with heme, we propose that the use of DCFH-DA could be useful in detection of the heme moiety of hemeprotein in vivo., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Naphthofluorescein and/or seminaphthofluorescein derivatives possessing the additional benzene units to one or both sides of fluorescein were exhaustively constructed through Friedel-Crafts type reactions between corresponding aroylbenzoic acids and dihydroxynaphthalenes. Compound 4 works as a one-dye pH indicator, which shows red in strong acid condition and blue in basic solution. Compound 23 (diacetate of compound 4) shows good transitivity to the HEK 293 cells and acts as a fluorescent pigment for the living cell imaging. Compounds 5, 6, and 9 show fluorescent emission in the NIR region (>700 nm) and imply the potentialities of NIR fluorescent probes.

Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix-loop-helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358-21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528-2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys-Fe-His and His-Fe-His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The thermodynamics of cofactor binding to the isolated reductase domain (Red) of nNOS and its mutants have been studied by isothermal titration calorimetry. The NADP(+) and 2',5'-ADP binding stoichiometry to Red were both 1:1, consistent with a one-site kinetic model instead of a two-site model. The binding constant (K(D) = 71 nM) and the large heat capacity change (ΔC(p) = -440 cal mol(-1) K(-1)) for 2',5'-ADP were remarkably different from those for NADP(+) (1.7 μM and -140 cal mol(-1) K(-1), respectively). These results indicate that the nicotinamide moiety as well as the adenosine moiety has an important role in binding to nNOS. They also suggest that the thermodynamics of the conformational change in Red caused by cofactor binding are significantly different from the conformational changes that occur in cytochrome c reductase, in which the nicotinamide moiety of the cofactor is not essential for binding. Analysis of the deletion mutant of the autoinhibitory helix (RedΔ40) revealed that the deletion resulted in a decrease in the binding affinity of 2',5'-ADP with more unfavorable enthalpy gain. In the case of RedCaM, which contains a calmodulin (CaM) binding site, the presence of Ca(2+)/CaM caused a 6.7-fold increase in the binding affinity for 2',5'-ADP that was mostly due to the favorable entropy change. These results are consistent with a model in which Ca(2+)/CaM induces a conformational change in NOS to a flexible "open" form from a "closed" form that locked by cofactor binding, and this change facilitates the electron transfer required for catalysis.

Crystal structures of nitric oxide synthases (NOS) isoforms have shown the presence of a strongly conserved heme active-site residue, Tyr588 (numbering for rat neuronal NOS, nNOS). Preliminary biochemical studies have highlighted its importance in the binding and oxidation to NO of natural substrates L-Arg and N(omega)-hydroxy-L-arginine (NOHA) and suggested its involvement in mechanism. We have used UV-visible and EPR spectroscopy to investigate the effects of the Tyr588 to Phe mutation on the heme-distal environment, on the binding of a large series of guanidines and N-hydroxyguanidines that differ from L-Arg and NOHA by the nature of their alkyl- or aryl-side chain, and on the abilities of wild type (WT) and mutant to oxidize these analogues with formation of NO. Our EPR experiments show that the heme environment of the Tyr588Phe mutant differs from that of WT nNOS. However, the addition of L-Arg to this mutant results in EPR spectra similar to that of WT nNOS. Tyr588Phe mutant binds L-Arg and NOHA with much weaker affinities than WT nNOS but both proteins bind non alpha-amino acid guanidines and N-hydroxyguanidines with close affinities. WT nNOS and mutant do not form NO from the tested guanidines but oxidize several N-hydroxyguanidines with formation of NO in almost identical rates. Our results show that the Tyr588Phe mutation induces structural modifications of the H-bonds network in the heme-distal site that alter the reactivity of the heme. They support recent spectroscopic and mechanistic studies that involve two distinct heme-based active species in the two steps of NOS mechanism., (Copyright 2010 Elsevier Inc. All rights reserved.)

The heme domain of neuronal PAS domain protein 2 (NPAS2), a transcription factor that regulates the mammalian circadian rhythm, has been suggested to act as a sensor for carbon monoxide. To characterize the role of the heme domain in this function, we investigated the effects of PASA domain mutants, in the context of full-length NPAS2, on the transcriptional activity of the mouse Period 1 gene in NIH3T3 cells. Mutation of the endogenous ligand for ferrous heme (H119A or H171A) resulted in remarkably reduced transcriptional activity. In gel-shift assays, H119A or H171A mutants of the isolated basic helix-loop-helix (bHLH)-PASA domain impaired heterodimer formation with BMAL1, resulting in loss of DNA binding to the canonical E-box (CACGTG). These results indicate that the transcriptional activities of the mutants correlated well with their DNA-binding activities, suggesting that local conformational changes near the axial ligands of the PASA domain are responsible for its regulation of transcription.

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix-loop-helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 x 10(7) mol(-1) x s(-1)) was more than two orders of magnitude higher than for association with apo-PAS-A (< 10(5) mol(-1) x s(-1)). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)-NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding.

Heme-regulated phosphodiesterase from Escherichia coli (DOS(Ec)) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOS(Ec) was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOS(Ec) using cAMP as a substrate is only 0.15 min(-1), which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOS(Ec) gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOS(Ec) gene knockout strain (Deltados). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOS(Ec) is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS(Ec) expression.

Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms. This protein forms a heterodimer with BMAL1, which binds to the E-box sequence to mediate circadian rhythm-regulated transcription. NPAS2 has two PAS domains with heme-binding sites in the N-terminal portion. In this study, we overexpressed wild-type and His mutants of the PAS-B domain (residues 241-416) of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins. Optical absorption spectra of the wild-type protein showed that the Fe(III), Fe(II) and Fe(II)-CO complexes are 6-co-ordinated low-spin complexes. On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordinated high-spin and 6-co-ordinated low-spin complexes. Based on inverse correlation between nu(Fe-CO) and nu(C-O) of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme-bound PAS-B is His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared. The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas, like the wild-type, the complexes for the five other His mutants were 6-co-ordinated low-spin complexes. Thus, our results suggest that one of the axial ligands of Fe(III) in PAS-B is His335. Also, binding kinetics suggest that heme binding to the PAS-B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin.

Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme-containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe3+-S stretching was observed at 334 cm(-1) for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys170 is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the nu11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c3, suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His119 and His171, but not Cys170, are axial ligands in the ferrous heme. This means that ligand replacement from Cys to His occurs upon heme reduction. The nu(Fe-CO) versus nu(C-O) correlation indicates that a neutral His is a trans ligand of CO. Our results support a mechanism in which CO binding disrupts the hydrogen bonding of His171 with surrounding amino acids, which induces conformational changes in the His171-Cys170 moiety, leading to physiological signaling.