Critical role of the heme axial ligand, Met95, in locking catalysis of the phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.     

Important roles of Tyr43 at the putative heme distal side in the oxygen recognition and stability of the Fe(II)-O2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.     

Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli

The heme-regulated phosphodiesterase from Escherichia coli (Ec DOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO, NO, and O2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain (Ec DosH) shows that Leu99, Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH and examined their physicochemical characteristics, including auto-oxidation rates, O2 and CO binding kinetics, and redox potentials. The Fe(III) complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants (0.049-0.33 min(-1)) were much higher than that of the wild-type (0.0063 min(-1)). Furthermore, the redox potentials of the former three mutants (23.1-34.6 mV versus SHE) were also significantly lower than that of the wild-type (63.9 mV versus SHE). Interaction between O2 and the L99F mutant was different from that in the wild-type, whereas CO binding rates of the mutants were similar to those of the wild-type. Thus, it appears that Leu99 and Leu115 are critical for determining the characteristics of heme iron. Finally, we discuss the roles of these amino-acid residues in the heme electronic states.     

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli. Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials.

On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.     

CO exchange of the oxyferrous complexes of endothelial nitric-oxide synthase oxygenase domain in the presence of 4-amino-tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a 1-electron donor to the oxyferrous-heme complex. 4-Amino-tetrahydrobiopterin (4-amino-BH4) inhibits NO synthesis, although it has similar redox properties. We recently reported that 4-amino-BH4 is capable of electron transfer to Fe(II).O(2) in cryogenic single-turnover [J. Biol. Chem. 278 (2003) 48602]. We also suggested that BH4 serves as a proton donor to the Fe(II).O(2)(-) complex, and that 4-amino-BH4 cannot perform this second essential function. To corroborate these claims and to further characterize the intermediates observed after oxygenation of NOS in the presence of 4-amino-BH4, we added CO immediately after O(2) addition to the reduced oxygenase domain of endothelial NOS at -30 degrees C. This resulted in complete formation of a P450-type Fe(II).CO complex with either Arg or NG-hydroxy-L-arginine as the substrate. In the presence of 4-amino-BH2, which is redox-inactive, the same procedure yielded ferric heme with either substrate, without formation of any Fe(II).CO complex. We conclude: (i) O(2) binding to ferrous heme in the presence of 4-amino-BH2 is essentially irreversible; (ii) 4-amino-BH4 can reduce the oxyferrous complex; (iii) O(2)(-), rather than H(2)O(2) is the immediate product of uncoupled catalysis in the presence of 4-amino-BH4.     

Roles of Arg-97 and Phe-113 in regulation of distal ligand binding to heme in the sensor domain of Ec DOS protein. Resonance Raman and mutation study

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O(2), CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 --> Ile, Met-95 --> Ala, Arg-97 --> Ile, Arg-97 --> Ala, Arg-97 --> Glu, Phe-113 --> Leu, and Phe-113 --> Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.     

Functional implications of the propionate 7-arginine 220 interaction in the FixLH oxygen sensor from Bradyrhizobium japonicum

BjFixL from Bradyrhizobium japonicum is a heme-based oxygen sensor implicated in the signaling cascade that enables the bacterium to adapt to fluctuating oxygen levels. Signal transduction is initiated by the binding of O(2) to the heme domain of BjFixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. We report structural changes of the heme and its binding pocket in the Fe(II) deoxy and Fe(III) met states of the wild-type BjFixLH oxygen sensor domain and four mutants of the highly conserved residue arginine 220. UV-visible, electron paramagnetic resonance, and resonance Raman spectroscopies all showed that the heme iron of the R220H mutant is unexpectedly six-coordinated at physiological pH in the Fe(III) state but undergoes pH- and redox-dependent coordination changes. This behavior is unprecedented for FixL proteins, but is reminiscent of another oxygen sensor from E. coli, EcDos. All mutants in their deoxy states are five-coordinated Fe(II), although we report rupture of the residue 220-propionate 7 interaction and structural modifications of the heme conformation as well as propionate geometry and flexibility. In this work, we conclude that part of the structural reorganization usually attributed to O(2) binding in the wild-type protein is in fact due to rupture of the Arg220-P7 interaction. Moreover, we correlate the structural modifications of the deoxy Fe(II) states with k(on) values and conclude that the Arg220-P7 interaction is responsible for the lower O(2) and CO k(on) values reported for the wild-type protein.     

Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli. Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and Met-95 mutants

The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis. The rate of O(2) association (k(on)) with full-length Ec DOS is extremely slow at 0.0019 microM(-1) s(-1), compared with >9.5 microM(-1) s(-1) for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method. This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain. Dissociation constants (K(d)) calculated from the kinetic parameters are 340 and 20 microm for the full-length wild-type enzyme and its isolated PAS domain, respectively. Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the k(on) value by more than 30-fold, and consequently, a decrease in the K(d) value to less than 1 microM. The k(on) value for CO binding to the full-length wild-type enzyme is also very low (0.00081 microM(-1) s(-1)). The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O(2). However, the K(d) values for CO are considerably lower than those for O(2).     

The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis

Ec DOS is a heme-based gas sensor enzyme that catalyzes conversion from cyclic-di-GMP to linear-di-GMP in response to gas molecules, such as oxygen, CO and NO. Ec DOS contains an N-terminal heme-binding PAS domain and C-terminal phosphodiesterase domain. Based on crystal structures of the isolated heme-binding domain, it is suggested that the FG loop is involved in intra-molecular signal transduction to the catalytic domain. We generated nine full-length proteins mutated at ionic and non-ionic polar residues between positions 83 and 96 corresponding to the F-helix and FG loop, and examined the heme binding properties, autoxidation rates, and catalytic activities of mutant proteins. N84A and R85A mutant proteins displayed lower heme binding affinities, consistent with the finding that Asn84 interacts with propionate of protoporphyrin IX, and Arg85 with Asp40 on the heme proximal side. Autoxidation rates (0.058-0.54 min⁻¹) of R91A, S96A and K89A/R91A/E93A mutant proteins were significantly higher than that (0.0053 min⁻¹) of wild-type protein, suggesting that these residues in the FG loop form heme distal architecture conferring stability to the Fe(II)-O₂ complex. Catalytic activities of N84A and R85A mutant proteins with low heme affinity were significantly higher than those of wild-type protein in the absence of gas molecules. Accordingly, we propose that loss of heme binding enhances basal catalysis without the gas molecule, consistent with previous reports on heme inhibition of Ec DOS catalysis.     

Unusual cyanide bindings to a heme-regulated phosphodiesterase from Escherichia coli: effect of Met95 mutations

In order to understand heme environment of a heme-regulated phosphodiesterase (Ec DOS), the binding behavior of cyanide to the Fe (III) complex was examined. Interestingly, the rate of cyanide binding to full-length Ec DOS was unusually slow with k(on)=0.0022mM(-1)s(-1), while the rate for the isolated heme domain of Ec DOS (0.045mM(-1)s(-1)) was 20-fold higher. Ala and Leu mutations at Met95, which has been suggested to be a heme axial ligand, increased the k(on) rate 11- and 8-fold, respectively, and dramatically decreased the cyanide dissociation rate from the isolated heme domain. His mutation at Met95, on the other hand, caused a 17-fold decrease in the k(on) value. We discuss the unusual cyanide binding behavior and the role of Met95 in controlling cyanide binding.     

Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5

Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O(2), and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O(2) binding site at the heme distal side. The O(2) dissociation rate constant, 0.10 s(-1), was substantially increased up to 8.0 s(-1) upon Y45L mutation. The resonance Raman frequencies representing ν(Fe-O2) (559 cm(-1)) and ν(O-O) (1149 cm(-1)) of the Fe(II)-O(2) complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (ν(Fe-O2), 557 cm(-1); ν(O-O), 1141 cm(-1)), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.     

Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.     

Differential effects of alkyl- and arylguanidines on the stability and reactivity of inducible NOS heme-dioxygen complexes

NO-Synthases are heme proteins that catalyze the oxidation of L-arginine into NO and L-citrulline. Some non-amino acid alkylguanidines may serve as substrates of inducible NOS (iNOS), while no NO* production is obtained from arylguanidines. All studied guanidines induce uncoupling between electrons transferred from the reductase domain and those required for NO formation. This uncoupling becomes critical with arylguanidines, leading to the exclusive formation of superoxide anion O2*- as well as hydrogen peroxide H2O2. To understand these different behaviors, we have conducted rapid scanning stopped-flow experiments with dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) to study, respectively, the (i) autoxidation and (ii) activation processes of heme ferrous-O2 complexes (Fe(II)O2) in the presence of eight alkyl- and arylguanidines. The Fe(II)O2 complex is more easily autooxidized by alkylguanidines (10-fold) and arylguanidines (100-fold) compared to L-arginine. In the presence of alkylguanidines and BH4, the oxygen-activation kinetics are very similar to those observed with L-arginine. Conversely, in the presence of arylguanidines, no Fe(II)O2 intermediate is detected. To understand such variations in reactivity and stability of Fe(II)O2 complex, we have characterized the effects of alkyl- and arylguanidines on Fe(II)O2 structure using the Fe(II)CO complex as a mimic. Resonance Raman and FTIR spectroscopies show that the two classes of guanidine derivatives induce different polar effects on Fe(II)CO environment. Our data suggest that the structure of the substituted guanidine can modulate the stability and the reactivity of heme-dioxygen complexes. We thus propose differential mechanisms for the electron- and proton-transfer steps in the NOS-dependent, oxygen-activation process, contingent upon whether alkyl- or arylguanidines are bound.     

Structure and ligand binding properties of leucine 29(B10) mutants of human myoglobin.

Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29 (B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta meso carbon of the heme group to the one pointing toward the alpha meso carbon atom, presumably filling the space left by removal of the delta 2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)----Ala and Leu104 (G5)----Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.     

Roles of the heme distal residues of FixL in O2 sensing: a single convergent structure of the heme moiety is relevant to the downregulation of kinase activity

FixL is a heme-based O(2) sensor, in which the autophosphorylation is regulated by the binding of exogenous ligands such as O(2) and CN(-). In this study, mutants of the heme distal Arg200, Arg208, Ile209, Ile210, and Arg214 residues of SmFixL were characterized biochemically and physicochemically, because it has been suggested that they are significant residues in ligand-linked kinase regulation. Measurements of the autoxidation rate, affinities, and kinetics of ligand binding revealed that all of the above residues are involved in stabilization of the O(2)-heme complex of FixL. However, Arg214 was found to be the only residue that is directly relevant to the ligand-dependent regulation of kinase activity. Although the wild type and R214K and R214Q mutants exhibited normal kinase regulation, R214A, R214M, R214H, and R214Y did not. (13)C and (15)N NMR analyses for (13)C(15)N(-) bound to the truncated heme domains of the Arg214 mutants indicated that, in the wild type and the foregoing two mutants, the heme moiety is present in a single conformation, but in the latter four, the conformations fluctuate possibly because of the lack of an interaction between the iron-bound ligand and residue 214. It is likely that such a rigid conformation of the ligand-bound form is important for the downregulation of histidine kinase activity. Furthermore, a comparison of the NMR data between the wild type and R214K and R214Q mutants suggests that a strong electrostatic interaction between residue 214 and the iron-bound ligand is not necessarily required for the single convergent structure and eventually for the downregulation of FixL.     

NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles

Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.