Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli

In the heme-based oxygen sensor Dos from Escherichia coli, one of the axial ligands (Met 95) of a six-coordinate heme can be replaced by external ligands such as O(2), NO, and CO, which causes a switch in phosphodiesterase activity. To gain insight into the bidirectional switching mechanism, we have studied the interaction of ligands with the sensor domain DosH by flash photolysis experiments with femtosecond time resolution. The internal ligand can be photodissociated from the ferrous heme and recombines with time constants of 7 and 35 ps. This is somewhat slower than recombination of the external ligands NO, with which picosecond rebinding occurs with unprecedented efficiency (>99%) with a predominant phase of approximately 5 ps, and O(2) (97% in 5 ps, Liebl, U., Bouzhir-Sima, L., Négrerie, M., Martin, J.-L., and Vos, M. H. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 12771-12776). Dissociated CO displays geminate rebinding in 1.5 ns with a very high yield (60%). Together these results indicate that the heme environment provides a very tight pocket for external ligands, presumably preventing frequent switching events. Additional CO dissociation and rebinding experiments on a longer time scale reveal that (a) Met 95 binding, in 100 micros, occurs in competition with bimolecular CO binding, and (b) subsequent replacement of Met 95 by CO on the millisecond time scale occurs faster than in rapid-mixing experiments, suggesting a slow further relaxation. A minimal ligand binding model is proposed that suggests that Met 95 displacement from the heme is facilitated by the presence of an external ligand in the heme environment. Furthermore, the orders of magnitude difference between Met 95 binding after dissociation of internal and external ligands, as well as the spectral characteristics of photodissociation intermediates, indicate substantial rearrangement of the heme environment associated with ligand sensing. Further remarkable observations include evidence for stable (>4 ns) photooxidation of six-coordinate ferrous heme, with a quantum yield of 4-8%.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Insights into signal transduction involving PAS domain oxygen-sensing heme proteins from the X-ray crystal structure of Escherichia coli Dos heme domain (Ec DosH)

The X-ray crystal structure of the Escherichia coli (Ec) direct oxygen sensor heme domain (Ec DosH) has been solved to 1.8 A using Fe multiple-wavelength anomalous dispersion (MAD), and the positions of Met95 have been confirmed by selenomethionine ((Se)Met) MAD. Ec DosH is the sensing part of a larger two-domain sensing/signaling protein, in which the signaling domain has phosphodiesterase activity. The asymmetric unit of the crystal lattice contains a dimer comprised of two differently ligated heme domain monomers. Except for the heme ligands, the monomer heme domains are identical. In one monomer, the heme is ligated by molecular oxygen (O(2)), while in the other monomer, an endogenous Met95 with S --> Fe ligation replaces the exogenous O(2) ligand. In both heme domains, the proximal ligand is His77. Analysis of these structures reveals sizable ligand-dependent conformational changes in the protein chain localized in the FG turn, the G(beta)-strand, and the HI turn. These changes provide insight to the mechanism of signal propagation within the heme domain following initiation due to O(2) dissociation.     

Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket.

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s(-1), is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5-10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.     

Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications.

The nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and NO through consumption of oxygen bound to the heme. Because NO is produced close to the heme and may bind to it, its subsequent role in a regulatory mechanism should be scrutinized. We therefore examined the kinetics of NO rebinding after photodissociation in the heme pocket of human endothelial NOS by means of time-resolved absorption spectroscopy. We show that geminate recombination of NO indeed occurs and that this process is strongly modulated by L-Arg. This NO rebinding occurs in a multiphasic fashion and spans over 3 orders of magnitude. In both ferric and ferrous states of the heme, a fast nonexponential picosecond geminate rebinding first takes place followed by a slower nanosecond phase. The rates of both phases decreased, whereas their relative amplitudes are changed by the presence of L-Arg; the overall effect is a slow down of NO rebinding. For the isolated oxygenase domain, the picosecond rate is unchanged, but the relative amplitude of the nanosecond binding decreased. We assigned the nanosecond kinetic component to the rebinding of NO that is still located in the protein core but not in the heme pocket. The implications for a mechanism of regulation involving NO binding are discussed.     

Primary processes in heme-based sensor proteins

A wide and still rapidly increasing range of heme-based sensor proteins has been discovered over the last two decades. At the molecular level, these proteins function as bistable switches in which the catalytic activity of an enzymatic domain is altered mostly by binding or dissociation of small gaseous ligands (O₂, NO or CO) to the heme in a sensor domain. The initial “signal” at the heme level is subsequently transmitted within the protein to the catalytic site, ultimately leading to adapted expression levels of specific proteins. Making use of the photolability of the heme-ligand bond that mimics thermal dissociation, early processes in this intra-protein signaling pathway can be followed using ultrafast optical spectroscopic techniques; they also occur on timescales accessible to molecular dynamics simulations. Experimental studies performed over the last decade on proteins including the sensors FixL (O₂), CooA (CO) and soluble guanylate cyclase (NO) are reviewed with an emphasis on emerging general mechanisms. After heme-ligand bond breaking, the ligand can escape from the heme pocket and eventually from the protein, or rebind directly to the heme. Remarkably, in all sensor proteins the rebinding, specifically of the sensed ligand, is highly efficient. This ”ligand trap” property possibly provides means to smoothen the effects of fast environmental fluctuations on the switching frequency. For 6-coordinate proteins, where exchange between an internal heme-bound residue and external gaseous ligands occurs, the study of early processes starting from the unliganded form indicates that mobility of the internal ligand may facilitate signal transfer. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

For many years, myoglobin has served as a paradigm for structure–function studies in proteins. Ligand binding and migration within myoglobin has been studied in great detail by crystallography and spectroscopy, showing that gaseous ligands such as O₂, CO, and NO not only bind to the heme iron but may also reside transiently in three internal ligand docking sites, the primary docking site B and secondary sites C and D. These sites affect ligand association and dissociation in specific ways. Neuroglobin is another vertebrate heme protein that also binds small ligands. Ligand migration pathways in neuroglobin have not yet been elucidated. Here, we have used Fourier transform infrared temperature derivative spectroscopy at cryogenic temperatures to compare the influence of the side chain volume of amino acid residue B10 on ligand migration to and rebinding from docking sites in myoglobin and neuroglobin.     

Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange

A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.     

A flash photolysis method to characterize hexacoordinate hemoglobin kinetics

A flash photolysis method is described for analyzing ligand binding to the new and growing group of hemoglobins which are hexacoordinate in the unligated, ferrous state. Simple analysis of a two exponential fit to time courses for CO rebinding at varying CO concentrations yields rate constants for formation and dissociation of the hexacoordinate complex, and the bimolecular rate constant for CO binding. This method was tested with a nonsymbiotic plant hemoglobin from rice for which these values had not previously been determined. For this protein, dissociation and rebinding of the hexacoordinating amino acid side chain, His(73), is rapid and similar to the rate of CO binding at high CO concentrations. These results indicate that hexacoordination must be taken into account when evaluating the affinity of hexacoordinate hemoglobins for ligands.     

Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: implications for nitric oxide sensors

The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases ( approximately 10 and approximately 100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes.     

The truncated oxygen-avid hemoglobin from Bacillus subtilis: X-ray structure and ligand binding properties

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 A resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 A).     

Viscosity-dependent relaxation significantly modulates the kinetics of CO recombination in the truncated hemoglobin TrHbN from Mycobacterium tuberculosis

Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)     

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.     

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.     

Altered ligand rebinding kinetics due to distal-side effects in hemoglobin chico (Lysbeta66(E10) --> thr)

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis.

Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R. E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a "control" analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket.