Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment (original) (raw)
Related papers
Biochemistry, 1999
The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a 3 oxoferryl (Fe 4+ dO 2-+ H 2 O). Since the conversion of ferrous to ferryl heme a 3 (Fe 2+ to Fe 4+) is sufficient for this reaction, presumably Cu B remains reduced in the product, along with Cu A and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a 3 oxoferryl is reduced to ferric hydroxide (Fe 3+ OH-). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu B is oxidized. This step has a time constant of about 65 µs. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu A must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 µs, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a 3 Fe 3+ OH-), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V max) 250 s-1 , K m) 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electronreduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a 3 ferric species. The reaction of hydrogen peroxide with heme a 3 is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.
Evidence for Heme Oxygenase Activity in a Heme Peroxidase
Biochemistry, 2009
The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H 2 O 2 -dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O 2 -dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the R-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism.
The time-resolved kinetics of substrate oxidation and cosubstrate H 2 O 2 reduction by dehaloperoxidase-hemoglobin (DHP) on a seconds-to-minutes time scale was analyzed for peroxidase substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol (2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate inhibition at high concentration due to the internal binding at the distal pocket of DHP, whereas ABTS does not show substrate inhibition at any concentration. The data are consistent with an external binding site for the substrates with an internal substrate inhibitor binding site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic behavior of horseradish peroxidase (HRP) in terms of k cat , K m AH 2 and K m H 2 O 2 using the same kinetic scheme. Unlike DHP, HRP does not exhibit any measurable substrate inhibition, consistent with substrate binding at the edge of heme near the protein surface at all substrate concentrations. The binding of substrates and their interactions with the heme iron were further compared between DHP and HRP using a competitive fluoride binding experiment, which provides a method for quantitative measurement of internal association constants associated with substrate inhibition. These experiments show the regulatory role of an internal substrate binding site in DHP from both a kinetic and competitive ligand binding perspective. The interaction of DHP with substrates as a result of internal binding actually stabilizes that protein and permits DHP to function under conditions that denature HRP. As a consequence, DHP is a tortoise, a slow but steady enzyme that wins the evolutionary race against the HRP-type of peroxidase, which is a hare, initially rapid, but flawed for this application because of the protein denaturation under the conditions of the experiment.
Structure and Reaction Mechanism in the Heme Dioxygenases
Biochemistry, 2011
T he heme dioxygenase enzymes catalyze the first and ratelimiting step in the kynurenine pathway, the O 2 -dependent oxidation of L-tryptophan to N-formylkynurenine. This reaction is unique in heme chemistry. Somewhat confusingly, this enzyme in the early literature is termed "tryptophan pyrrolase" 1À3 (and even "tryptophan peroxidase-oxidase" 4 ); only later did the nomenclature converge on either tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Actually, both TDO and IDO catalyze the same reaction (Scheme 1), with the different denominations merely reflecting the generally wider substrate specificity of the IDOs compared to the more substratespecific TDOs.
Biochemistry, 2008
In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows:-481 mV (WT),-477 mV (A320Q),-453 mV (F310A), and-450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H 2 O 2 show the following trend: WT ≈ A320Q < F310A ≈ F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H 2 O 2 .
A catalytic approach to estimate the redox potential of heme-peroxidases
Biochemical and Biophysical Research Communications, 2007
The redox potential of heme-peroxidases varies according to a combination of structural components within the active site and its vicinities. For each peroxidase, this redox potential imposes a thermodynamic threshold to the range of oxidizable substrates. However, the instability of enzymatic intermediates during the catalytic cycle precludes the use of direct voltammetry to measure the redox potential of most peroxidases. Here we describe a novel approach to estimate the redox potential of peroxidases, which directly depends on the catalytic performance of the activated enzyme. Selected p-substituted phenols are used as substrates for the estimations. The results obtained with this catalytic approach correlate well with the oxidative capacity predicted by the redox potential of the Fe(III)/Fe(II) couple.
Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase
Journal of Biological Chemistry, 2011
An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (␦ ؍ 0.055 mm/s and ⌬E Q ؍ 1.755 mm/s) and a protein-based free radical (g ؍ 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation byproducts by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.