Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from Thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a(3), and for Glu126(II) (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126(II), and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a(3), was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a(3) or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a(3) is a good candidate to be the proton loading site.     

Mechanisms for the oxidation of reduced gluthathione by stimulated granulocytes

We have reported previously that human granulocytes have an irreversible fall in their endogenous reduced soluble sulfhydryls following zymosan stimulation. In the present study, we demonstrate that stimulated granulocytes release one or more reactive oxygen species (ROS) with the capacity to oxidize reduced glutathione (GSH). One or more of these compounds is stable enough to be detected in the supernatant. The formation of these stable oxidants appears to require H2O2 and heme or a heme-containing enzyme. However, once formed, the compound reacts with GSH without these factors. The ROS is not superoxide or hydroxyl radical, since neither superoxide dismutase nor the hydroxyl scavengers, mannitol and benzoic acid, change the rate of the reaction. Methionine has recently been demonstrated to be oxidized to a sulfoxide by a reactive oxygen species that is dependent on H2O2 and heme for its production. We found that methionine could directly react with the same ROS that degrades GSH. The ROS also has the capacity to oxidize iodide and fix halogen to proteins. Our data indicate that stimulated granulocytes release a ROS with the capacity to oxidize GSH, react with methionine, and oxidize and fix I- to protein. The compound, therefore, appears dependent on H2O2 and the myeloperoxidase system for its production, and is either hypochlorous acid (HOCI) or a compound derived from HOCI, such as a chloramine. The capacity of GSH to react with this ROS suggests an additional role for this tripeptide in cellular protection against oxidant injury.     

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the "peroxy" state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH approximately 7.5. The movement of the Lys is proposed to regulate proton transfer by "shutting off" the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This "shutoff" prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.     

Proposed Conformation of Oxytocin in Solution

A conformation of the neurohypophyseal hormone oxytocin in solution is proposed. The structure possesses, in addition to the beta-turn comprised of the sequence -L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl- in the ring component of the hormonal molecule, a second beta-turn involving the C-terminal oxytocin sequence, -L-cysteinyl-L-prolyl-L-leucylglycinamide. The resulting oxytocin structure places the bulky side chains of the leucine and isoleucine residues, as well as the cyclic moiety of the proline residue, at corners of the two beta-turns. A critical role is played by the asparagine residue: its peptide N-H participates in the formation of the hydrogen-bonded cyclic structure of the beta-turn in the ring component of oxytocin and its peptide C=O can be hydrogen-bonded to the N-H of tyrosine, while its side chain C=O stabilizes the second beta-turn by forming a hydrogen bond with the N-H of the leucine residue, which is part of the end peptide of the second beta-turn. This conformational assignment of oxytocin is consistent with hydrogen-deuterium exchange studies, with plots of temperature dependence of peptide proton chemical shifts, and with the coupling constants for the NH-CH dihedral angles.     

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.     

Mechanism of and exquisite selectivity for O-O bond formation by the heme-dependent chlorite dismutase

Chlorite dismutase (Cld) is a heme b-dependent, O–O bond forming enzyme that transforms toxic chlorite (ClO₂⁻) into innocuous chloride and molecular oxygen. The mechanism and specificity of the reaction with chlorite and alternate oxidants were investigated. Chlorite is the sole source of dioxygen as determined by oxygen-18 labeling studies. Based on ion chromatography and mass spectrometry results, Cld is highly specific for the dismutation of chlorite to chloride and dioxygen with no other side products. Cld does not use chlorite as an oxidant for oxygen atom transfer and halogenation reactions (using cosubstrates guaiacol, thioanisole, and monochlorodimedone, respectively). When peracetic acid or H₂O₂ was used as an alternative oxidant, oxidation and oxygen atom transfer but not halogenation reactions occurred. Monitoring the reaction of Cld with peracetic acid by rapid-mixing UV-visible spectroscopy, the formation of the high valent compound I intermediate, [(Por^•+)Fe^IV = O], was observed [k₁ = (1.28 ± 0.04) × 10⁶ M⁻¹ s⁻¹]. Compound I readily decayed to form compound II in a manner that is independent of peracetic acid concentration (k₂ = 170 ± 20 s⁻¹). Both compound I and a compound II-associated tryptophanyl radical that resembles cytochrome c peroxidase (Ccp) compound I were observed by EPR under freeze-quench conditions. The data collectively suggest an O–O bond-forming mechanism involving generation of a compound I intermediate via oxygen atom transfer from chlorite, and subsequent recombination of the resulting hypochlorite and compound I.     

The proton donor for O-O bond scission by cytochrome c oxidase

Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate P(R) of the binuclear active site composed of heme a(3) and Cu(B). The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu(B). To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the P(R) intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximately 60 micros) appearance of the P(R) species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the P(R) species suggests transfer of a proton over a distance of approximately 4 A perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O(2) reduction in conditions where heme a is oxidized.     

Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide

We report direct evidence for the existence of an iron(IV)-hydroxide. Resonance Raman measurements on chloroperoxidase compound II (CPO-II) reveal an isotope ((18)O and (2)H)-sensitive band at nu(Fe-O) = 565 cm(-1). Preparation of CPO-II in H(2)O using H(2)(18)O(2) results in a red-shift of 22 cm(-1), while preparation of CPO-II in (2)H(2)O using H(2)O(2) results in a red-shift of 13 cm(-1). These values are in good agreement with the isotopic shifts predicted (23 and 12 cm(-1), respectively) for an Fe-OH harmonic oscillator. The measured Fe-O stretching frequency is also in good agreement with the 1.82-A Fe-O bond reported for CPO-II. A Badger's rule analysis of this distance provides an Fe-O stretching frequency of nu(Badger) = 563 cm(-1). We also present X-band electron nuclear double resonance (ENDOR) data for cryoreduced CPO-II. Cryogenic reduction (77 K) of the EPR-silent Fe(IV)OH center in CPO-II results in an EPR-active Fe(III)OH species with a strongly coupled (13.4 MHz) exchangeable proton. Based on comparisons with alkaline myoglobin, we assign this resonance to the hydroxide proton of cryoreduced CPO-II.     

Verdohemochrome IX alpha: preparation and oxidoreductive cleavage to biliverdin IX alpha.

Several studies have shown that both terminal oxygen atoms of biliverdin are derived from molecular oxygen. Since the conversion of verdohemochrome to biliverdin has been assumed to be hydrolytic, these findings seemed to exclude verdohemochrome as an intermediate in the degradation of heme to biliverdin. Coupled oxidation of myoglobin and ascorbate yielded a pure preparation of verdohemochrome IX alpha. The structure and ferrous state of this product were determined from its composition, ligand reactions, 1H NMR spectrum, and conversion to biliverdin IX alpha dimethyl ester. Reaction with ascorbate and 18O2 converted this compound to biliverdin that contained an atom of 18O. Successive treatment of verdohemochrome, first oxidation with H2O2 and then reduction with phenylhydrazine, yielded the iron complex of biliverdin. These results showed that hydrolysis is not an obligatory step in the conversion of verdohemochrome to biliverdin and, moreover, indicated how heme can be converted, with verdohemochrome as an intermediate, into biliverdin in which the two terminal oxygen atoms are derived from different O2 molecules.     

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.     

Theoretical studies on pro-leu-gly-nh2 conformation.

Classical potential function calculations were carried out on the hypothalamic factor Pro-Leu-Gly-NH2. The results indicate that the proposed 10-membered, hydrogen-bonded beta-turn conformation of this tripeptide is a strongly preferred structure. Its stability appears to be inherent in the rather rigid backbone conformation of the leucine residue rather than the hydrogen bond between the carboxamide proton of glycinamide and the C=O of the proline moiety; the glycinamide has little influence on the phi-psi of the leucine backbone structure. The type II beta-turn structure of the Pro-Leu-Gly-NH2 is preferred.     

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

Recently, we discovered an intriguing hemoprotein [aliphatic aldoxime dehydratase (OxdA)] that catalyzes the dehydration of aliphatic aldoximes [R-CH=N-OH] to the corresponding nitriles [R-C identical withN] in the industrial Pseudomonas chlororaphis B23 strain. Unlike the utilization of H(2)O(2) or O(2) as a mediator of the catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron, experimental evidence of which was obtained here by means of resonance Raman (RR) analysis with an isotope technique. We found that the addition of a large amount of butyraldoxime (final concentration, 200 mM) to ferrous OxdA with a low enzyme concentration (final concentration, 5 muM) yields a long-lived OxdA-substrate complex (named OS-II), whose UV-vis spectrum is different from the corresponding spectra of the OxdA-substrate complex I and CO-bound, ferrous, and ferric forms of OxdA. Intriguingly, the RR analysis demonstrated that OS-II includes a highly oxidized heme with strong bonding between a substrate and the heme iron, as judged from the heme oxidation state marker nu(4) band at 1,379 cm(-1) and the (15)N-isotope-substituted butyraldoxime sensitive band at 857 cm(-1) in the RR spectra. It is noteworthy that OS-II has a highly oxidized heme like the ferryl-oxo heme species (e.g., compound II) formed by some general hemoproteins, although the function of OxdA is different from those (transport of electrons, transport of oxygen, sensing of oxygen or carbon monoxide, and catalysis of redox reactions) of general hemoproteins.     

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located approximately 30 A from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from approximately 9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P --> F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.     

Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27 degrees C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H(2)O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H(2)O) as a function of oxygenation, the amount of oxygen bound to the alpha and beta chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential O(2) binding to the alpha or beta chains; (ii) in the presence of organic phosphates, the alpha hemes have a higher affinity for O(2) as compared to the beta hemes; (iii) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (iv) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.     

Water surface is acidic

Water autoionization reaction 2H2O --> H3O- + OH- is a textbook process of basic importance, resulting in pH = 7 for pure water. However, pH of pure water surface is shown to be significantly lower, the reduction being caused by proton stabilization at the surface. The evidence presented here includes ab initio and classical molecular dynamics simulations of water slabs with solvated H3O+ and OH- ions, density functional studies of (H2O)(48)H+ clusters, and spectroscopic isotopic-exchange data for D2O substitutional impurities at the surface and in the interior of ice nanocrystals. Because H3O+ does, but OH- does not, display preference for surface sites, the H2O surface is predicted to be acidic with pH < 4.8. For similar reasons, the strength of some weak acids, such as carbonic acid, is expected to increase at the surface. Enhanced surface acidity can have a significant impact on aqueous surface chemistry, e.g., in the atmosphere.     

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.     

Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.     

Gating of proton and water transfer in the respiratory enzyme cytochrome c oxidase

The membrane-bound enzyme cytochrome c oxidase is responsible for cell respiration in aerobic organisms and conserves free energy from O2 reduction into an electrochemical proton gradient by coupling the redox reaction to proton-pumping across the membrane. O2 reduction produces water at the bimetallic heme a3/CuB active site next to a hydrophobic cavity deep within the membrane. Water molecules in this cavity have been suggested to play an important role in the proton-pumping mechanism. Here, we show by molecular dynamics simulations that the conserved arginine/heme a3 delta-propionate ion pair provides a gate, which exhibits reversible thermal opening that is governed by the redox state and the water molecules in the cavity. An important role of this gate in the proton-pumping mechanism is supported by site-directed mutagenesis experiments. Transport of the product water out of the enzyme must be rigidly controlled to prevent water-mediated proton leaks that could compromise the proton-pumping function. Exit of product water is observed through the same arginine/propionate gate, which provides an explanation for the observed extraordinary spatial specificity of water expulsion from the enzyme.     

Role of myeloperoxidase in the respiratory burst of human neutrophils

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.     

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.