PAS domains, which have been identified in over 1100 proteins from all three kingdoms of life, convert various input stimuli into signals that propagate to downstream components by modifying protein-protein interactions. One such protein is the Escherichia coli redox sensor, Ec DOS, a phosphodiesterase that degrades cyclic adenosine monophosphate in a redox-dependent manner. Here we report the crystal structures of the heme PAS domain of Ec DOS in both inactive Fe(3+) and active Fe(2+) forms at 1.32 and 1.9 A resolution, respectively. The protein folds into a characteristic PAS domain structure and forms a homodimer. In the Fe(3+) form, the heme iron is ligated to a His-77 side chain and a water molecule. Heme iron reduction is accompanied by heme-ligand switching from the water molecule to a side chain of Met-95 from the FG loop. Concomitantly, the flexible FG loop is significantly rigidified, along with a change in the hydrogen bonding pattern and rotation of subunits relative to each other. The present data led us to propose a novel redox-regulated molecular switch in which local heme-ligand switching may trigger a global "scissor-type" subunit movement that facilitates catalytic control.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.

Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the

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.

The heme environments of Met 95 and His 77 mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band ( Fe-His ) at 214 cm ؊1 , and the Fe-CO versus CO plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the Fe-His band but gave a Fe-Im band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a fivecoordinate heme complex. Taken Heme-containing signal-transducing proteins (1-3) respond to diatomic molecules, which act as physiological, environmental messengers. This has attracted the attention of biophysical chemists. The O 2 sensing proteins so far identified include FixL (an oxygen-sensing kinase of Rhizobia meliloti) (1, 4), HemAT (an oxygen sensor heme protein discovered from Bacillus subtilis (HemAT-Bs) and Halobacterium salinarium (HemAT-Hs)) (5, 6), PDEA1 (7), and putatively a heme protein from E. coli (designated Escherichia coli DOS) (8). There is only one CO sensor protein known (CooA, a CO-binding transcriptional regulation factor from Rhodospirillum rubrum) (9, 10) and one NO sensor (soluble guanylate cyclase) (11,12). In each case, binding of an external ligand to the heme located in an N-terminal sensory domain transmits a signal to the functional C-terminal domain (either enzymatic or DNA binding). We are curious to know how these proteins recognize a specific diatomic molecule to generate the appropriate physiological response and what kind of structural changes occur to transmit the signal from the sensory domain to the functional domain.The sensory domain of FixL belongs to the large family of signal-transducing PAS domain 1 proteins, whereas those of HemAT, CooA, and soluble guanylate cyclase do not. The PAS domain proteins found in eukarya, archaea, and bacteria contain a partly conserved tertiary structure despite their limited sequence homology (Ͻ15%) and dissimilar cofactors (13). Although structures of three PAS proteins including the human voltage sensor (HERG) (14), the rhizobial oxygen sensor (FixL) (15, 16), and bacterial light sensor (PYP) (17) have been solved, interactions between the sensory domain and the functional domain are not clearly understood. Namely, hydrophobic interactions seem important to regulate the K ϩ channel of HERG, whereas polar interactions in the EF loop of the PAS domain seem to be essential to PYP. In the case of FixL, either a protein conformational change associated with the location of the heme iron (in-pla...

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and a-actin genes. In contrast, the simian virus 40 early genes, the 3-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-H factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.The interactions between specific DNA sequences, regulatory factors, and RNA polymerase II play an important role in modulating gene expression at the level of transcription. Many studies with DNA templates containing specific mutations have identified the cis-acting elements in DNA such as the conserved TATA box and the regions further upstream which are necessary for the efficient and accurate initiation of transcription. Recently, the GGGCGG sequence (GC box) located upstream of various genes was found to be important for their expression since deletion or mutation of this sequence resulted in a reduction or inactivation of their expression (14,22,23). Furthermore, Tjian and co-workers (7-10, 16, 19) have isolated a protein factor, SP1, which binds to the GC box and is essential for the transcription of this class of genes. Similarly, the CAAT box sequence (GGTCAATCT consensus) which is cis linked and located around 80 base pairs upstream from the RNA initiation site of several genes (1, 11) has also been found to be important for its expression (4, 6, 18). In this case, a protein binding to that region has been identified in our laboratory and others, but its function has not been defined (12,17,27).We have recently demonstrated that the distal promoter region of the chicken ovalbumin gene contains a duplicate GTCAAA box (GGTGTCAAAGGTCAAACT) which is essential fo...

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 Fe 3؉ -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 Cys 170 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 11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c 3 , 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 His In recent years, a variety of heme-containing gas sensor proteins have been discovered in different species, from bacteria to mammals (1-3). In these proteins, a change in the concentration of gas molecules such as NO, O 2 , or CO is detected by a heme group and transduced to the functional domain as a signal, leading to modulation of protein activity. The hemebased gas sensor proteins discovered so far are listed in Table I. NO is a signaling molecule involved in vasodilation and neuronal transmission (4, 5). Soluble guanylate cyclase (sGC) 1 is a well known heme-based NO sensor protein. Upon binding of NO to sGC, the iron-histidine bond in the N-terminal region, the sole covalent linkage between the heme and protein, is cleaved. The bond cleavage induces conformational changes, resulting in a 400-fold increase in GC activity in the C-terminal region (6). Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) also forms a five-coordinated NO-heme complex via Fe-His bond cleavage, resulting in activation by NO (7). The O 2 -sensing proteins identified so far include FixL (8), DOS (9), PDEA1 (2), and HemAT (10, 11). The sensory domains of FixL and Ec DOS belong to the PAS 2 superfamily. FixL is a hemebased oxygen sensor involved in the regulation of expression of nitrogen fixation genes in response to O 2 concentration (8, 12, 13). Under low O 2 concentrations, FixL is autophosphorylated at a histidine residue and transfers it to FixJ, whereas a high concentration of O 2 suppresses kinase activity (8). Ec DOS is also an O 2 (and/or redox) sensor protein identified in Escherichia coli that exhibits phosphodiesterase activity in an O 2 -dependent (and/or redox-dependent) manner (9, 14). CooA was the first CO sensor protein identified from a purple nonsulfur photosynthetic bacterium, Rhodospirillum rubrum (15, 16). When CO binds to heme, the accompanying conformational changes induce binding of CooA to its target DNA ...