Distal Pocket Polarity in the Unusual Ligand Binding Site of Soluble Guanylate Cyclase:  Implications for the Control of <sup>•</sup>NO Binding

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (ϳ60 cm ؊1 ) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm ؊1 , for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm ؊1 , respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm ؊1 , respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.It is now well recognized that hemoglobins (Hbs) 3 are widespread in all phyla. There has been a renewed interest in understanding the structure and function of Hbs in recent years (1-5), because these Hbs are located in diverse cellular compartments and possess atypical physicochemical properties. Hbs have been discovered in about 33% of the animal species, and in addition to being present in vertebrates, they are found in the phyla that include nematode, mollusc, and annelid, to name but a few. Single subunit globins have been discovered lately in algae, nonsymbiotic plants, and a number of prokaryotes ranging from bacteria to cyanobacteria (see Refs. 2-5). Additionally, flavoproteinbound chimeric Hbs have been found in bacteria (6) and yeast (7). In most cases, the nonvertebrate Hbs are proposed to have dissimilar functions from those in vertebrate Hbs; the latter class is established as consisting of O 2 carrier proteins. However, additional cellular activities have been proposed recently for vertebrate Hbs as well (8).Although the nonvertebrate Hbs bind O 2 and other ligands just as the vertebrate Hbs, the kinetic and structural properties of the O 2 complexes (see for example Refs...

Section: Discussionmentioning

confidence: 98%

Multiple Active Site Conformers in the Carbon Monoxide Complexes of Trematode Hemoglobins

Das

Dewilde

Friedman

et al. 2006

“…The YC-1-induced changes in the iron-CO stretching vibration ( Fe-CO ) have been reported (22), but precise analyses of the C-O stretching vibration ( C-O ) and of the effects of YC-1 on the C-O were not carried out (22,31). The infrared spectra of the 12 C 16 O complex under various conditions are summarized in Fig.…”

Section: Epr Characterization Of Five-and Six-coordinate Nohemes-thementioning

confidence: 99%

YC-1 Facilitates Release of the Proximal His Residue in the NO and CO Complexes of Soluble Guanylate Cyclase

Makino¹,

Obayashi²,

Homma³

et al. 2003

The benzylindazole compound YC-1 has been shown to activate soluble guanylate cyclase by increasing the sensitivity toward NO and CO. Here we report the action of YC-1 on the coordination of CO-and NO-hemes in the enzyme and correlate the events with the activation of enzyme catalysis. A single YC-1-binding site on the heterodimeric enzyme was identified by equilibrium dialysis. To explore the affect of YC-1 on the NO-heme coordination, the six-coordinate NO complex of the enzyme was stabilized by dibromodeuteroheme substitution. Using the dibromodeuteroheme enzyme, YC-1 converted the six-coordinate NO-heme to a five-coordinate NOheme with a characteristic EPR signal that differed from that in the absence of YC-1. These results revealed that YC-1 facilitated cleavage of the proximal His-iron bond and caused geometrical distortion of the five-coordinate NO-heme. Resonance Raman studies demonstrated the presence of two iron-CO stretch modes at 488 and 521 cm ؊1 specific to the YC-1-bound CO complex of the native enzyme. Together with the infrared C-O stretching measurements, we assigned the 488-cm ؊1 band to the iron-CO stretch of a six-coordinate CO-heme and the 521-cm ؊1 band to the iron-CO stretch of a fivecoordinate CO-heme. These results indicate that YC-1 stimulates enzyme activity by weakening or cleaving the proximal His-iron bond in the CO complex as well as the NO complex.

“…3a. In Table III, the observed frequencies of the major bands are compared with those of Mb (32,33), heme-bound heme oxygenase (32,33), and gas-sensing hemoproteins such as the O 2 , CO, and NO sensors of FixL (34 -37), CooA (38 -40), and sGC (41)(42)(43), respectively.…”

Section: Fig 1 Sds-page Of Purified Hemat-bs (A) and Estimation Of mentioning

confidence: 99%

“…In HemAT-Bs, however, the residue at the B10 position would be a leucine, an amino acid not capable of hydrogen bonding, as is the case for Sw Mb. The distal residue at the E7 position in Sw Mb and Pc Hb, His 64 and Gln 42 , respectively, also hydrogen bonds to the bound oxygen (23,78). In HemAT-Bs, the residue at the E7 position would be a serine, Ser 87 .…”

Section: Porphyrin Skeletal Vibrational Modes In the Low Frequency Rementioning

confidence: 99%

Resonance Raman and Ligand Binding Studies of the Oxygen-sensing Signal Transducer Protein HemAT from Bacillus subtilis

Aono¹,

Kato²,

Matsuki³

et al. 2002

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k on ‫؍‬ 32 M ؊1 s ؊1 and k off ‫؍‬ 23 s ؊1 , respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37°C was 0.06 h ؊1 , which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the Fe-O 2 band at a noticeably low frequency (560 cm ؊1 ), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the Fe-His band at a higher frequency (225 cm ؊1 ) than those of ordinary His-coordinated deoxy heme proteins. CObound HemAT-Bs gave the Fe-CO and C-O bands at 494 and 1964 cm ؊1 , respectively, which fall on the same C-O versus Fe-CO correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.Motile bacteria are known to swim toward or away from specific environmental stimuli such as nutrients, oxygen, or light (1, 2). This behavior, termed chemotaxis, is mediated by a signal transduction system consisting of methyl-accepting chemotaxis proteins (MCPs), 1 a histidine kinase CheA, a response regulator CheY, a coupling protein CheW, and the two enzymes that mediate sensory adaptation by covalently modifying the MCPs, CheR and CheB (3-6). Typical MCP is an integral membrane protein with an N-terminal periplasmic substrate binding domain and a C-terminal cytoplasmic signaling domain (7,8). The binding of a substrate, a repellent, or attractant to the periplasmic substrate binding domain is believed to induce a change in the MCP conformation that allows it to activate CheA (7, 8). The activated CheA phosphorylates CheY, and, consequently, the phosphorylated CheY binds to the switch complex at the base of the flagella to control the direction of flagellar rotation (3-6). In this signal transduction system, MCP acts as a sensor for the external signal and as a signal transducer.Hou et al. (9) have recently reported that Bacillus subtilis and Halobacterium salinarum have a signal transducer protein, HemAT-Bs and HemAT-Hs, respectively, for aerotaxis, the migratory response toward or away from oxygen. HemAT-Bs and HemAT-Hs are soluble proteins, and their C-terminal regions, residues 222-489 of HemAT-Hs and 198 -432 of HemAT-Bs, are 30% identical to the cytoplasmic signaling domain of Tsr, an MCP from Escherichia coli (9). Their N-terminal regions, residues 1-184 in HemAT-Hs and 1-175 in HemAT-Bs, show limited homology to myoglobin (9). Recombinant HemAT-Bs and HemAT-Hs are hemoproteins containing a b-type heme as a prost...