Ferric Cytochrome Research Articles

Photodissociation of Heme Distal Methionine in Ferrous Cytochrome c Revealed by Subpicosecond Time-Resolved Resonance Raman Spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7–9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV–visible (UV–vis) absorption and resonance Raman (RR). In 7–9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis–His-ligated heme center. In acidic 9 M urea solutions the UV–vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H 2O complex. The p K a for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the p K a 4.8. The formal redox potential of the His/H 2O complex of cytochrome c in 9 M urea at pH 3 was estimated to be −0.13 V, ca. 100 mV more positive than E°′ estimated for the bis–His complex of cytochrome c in urea solution at pH 7.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

Ferricytochrome c encapsulated in silica nanoparticles: structural stability and functional properties.

Using a modified sol-gel technique, we have succeeded in encapsulating ferric cytochrome c in silica nanoparticles obtained from hydrolysis and polycondensation of tetramethylorthosilicate. Particles dimensions have been determined with dynamic light scattering; this technique yields an hydrodynamic radius of about 100 nm, each nanoparticle containing about 10(2)-10(3) proteins. If stored in the cold at low ionic strength, nanoparticles are stable for more than one week, even if a slow radius increase with time is observed. CD measurements show that encapsulated proteins exhibit substantially increased stability against guanidinium hydrochloride induced denaturation. Reduction kinetics of encapsulated ferric cytochrome c by sodium dithionite, measured with standard stopped flow techniques, are slower by a factor of ten with respect to those measured in solution. Analogous experiments with myoglobin suggest that this slowing down is due to the diffusion time of dithionite within the silica matrix. Indeed, if a smaller ligand like CO is used, the intrinsic kinetic properties of encapsulated proteins are found to be unaltered even in the millisecond time range. The reported data show that our nanoparticles are extremely useful both for basic research, to study the stability and functions of encapsulated proteins, and for their potential biotechnological applications.

Substrate binding favors enhanced NO binding to P450cam.

Ferric cytochrome P450cam from Pseudomonas putida (P450cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO). The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly. In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate. The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure. Large and positive values of S and V observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO. In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process. Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level. Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects. The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.

Peripheral and Integral Binding of Cytochrome c to Phospholipids Vesicles

The interactions of ferric cytochrome c (Cyt-c) with dioleoyl-phosphatidylglycerol (DOPG) at low ionic strength have been studied by viscosity and turbidity measurements as well as by resonance Raman, circular dichroism, and UV−vis-absorption spectroscopy to monitor the structural changes of the liposomes and the protein upon complex formation. The observed mutual structural changes in the liposomes and the protein are associated with three different modes of protein binding. At high lipid/protein (L/P) ratios, Cyt-c binds electrostatically to the anionic headgroups of the phospholipids which induces structural changes of the protein. Decreasing the L/P-ratio weakens the electrostatic interactions such that membrane anchoring of Cyt-c can effectively compete with peripheral binding. This mode of binding is accompanied by an increase of long-range liposome−liposome interactions. Upon lowering the L/P-ratio below the ratio for full protein coverage of the vesicles, further Cyt-c binding is achieved via interactions with the protein monolayer. This mode of binding initiates phase separation of the liposome aggregates from the aqueous buffer. Our results indicate that the crucial parameter controlling the interplay between the binding modes appears to be the membrane surface potential which in turn sensitively depends on the protein coverage.

Electrochemical Control of Protein Monolayers at Indium Tin Oxide Surfaces for the Reagentless Optical Biosensing of Nitric Oxide

Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.

Mechanism of peroxynitrite interaction with cytochrome c.

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.

Mechanism of peroxynitrite interaction with cytochrome c.

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.

A Novel Method for Study of Protein Folding Kinetics by Monitoring Diffusion Coefficient in Time Domain

Molecular diffusion process after the photo-induced electron injection to ferric cytochrome c (Fe(III) cyt c) in guanidine hydrochloride (GdnHCl) 3.5M buffer solution is studied by the time-resolved transient grating technique. Circular dichroism studies have revealed that Fe(III) cyt c is unfolded under this condition but the reduced form, Fe(II) cyt c, is folded. Hence, this pulsed laser-induced reduction should initiate the folding process of cyt c. The observed transient grating signal shows prominent features, which have never been observed before. Based on several characteristic points, we concluded that the apparent diffusion coefficient (D) of Fe(II) cyt c after the reduction is time dependent, which must be associated with the protein folding dynamics. This time-dependent apparent D should reflect either the continuous time development of the hydrodynamic radius or population change of the unfolded and folded states during the folding dynamics. This is the first observation of the time-dependent apparent D during any chemical reaction, and this time-dependent measurement of D should be a unique and powerful way to study the protein folding kinetics from a viewpoint of the protein’s shape or the protein-water intermolecular interaction.

Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.

Resonance Raman investigations of cytochrome c conformational change upon interaction with the membranes of intact and Ca2+-exposed mitochondria.

The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.

Electrogeneration of Superoxide Ion and Its Mechanism at Thiol-Modified Au Electrodes in Alkaline Aqueous Solution

Electrogeneration of superoxide ion (O2−) via one-electron reduction of dioxygen (O2) in aqueous solution has been examined with various thiol-modified Au electrodes. Based on the reactions between O2− and superoxide dismutase (SOD) or ferric cytochrome c (cyt.c (Fe3+)), thiols (thiol compounds) such as n-hexadecanethiol, n-decanethiol, mercaptoacetic acid and 3-mercaptopropionic acid were found to be new electrode modifiers which allow the electrogeneration of O2− on Au electrode. The current efficiencies for the electrogeneration of O2− at these thiol-modified Au electrodes were ca. 4%. Some probable mechanisms for the electrogeneration of O2− at the modified electrodes are discussed. It is concluded that, unlike quinoline compounds adsorbed on Hg electrode which are known to form liquid-like hydrophobic layers favorable for the electrogeneration of O2−, the thiols adsorbing as self-assembled monolayers on the Au electrode surface do not necessarily form such hydrophobic layers, but prevent or reduce the spontaneous dismutation between O2 molecules adsorbing on the Au electrode surface by spatially separating them each other as a result of the adsorption of the thiol molecules themselves on it and consequently the electrogeneration of O2 can be realized at these thiol-modified Au electrodes.

Neutral thiol as a proximal ligand to ferrous heme iron: implications for heme proteins that lose cysteine thiolate ligation on reduction.

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O(2). A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175CD235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.

Mechanism and biological role of nitric oxide binding to cytochrome c'.

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.

Oxidation-state-dependent protein docking between cytochrome c and cytochrome b(5): high-pressure laser flash photolysis study.

To characterize the protein-protein interaction during electron transfer, we used Zn-substituted cytochrome c (ZnCytc) as a model of ferrous Cytc and determined the volume change, DeltaV(d)(Zn), for the dissociation of its complex with ferric cytochrome b(5) (Cytb(5)) by the pressure dependence of its photoinduced electron-transfer kinetics. Under ambient pressure, the dissociation constant, K(d)(Zn), of the ZnCytc/Cytb(5) complex was dependent on the buffer concentration, 1.5 and 12 microM in 2 and 10 mM Tris-HCl, pH 7.4, respectively, which was consistent with formation of salt bridges in its complexation. The dissociation of one salt bridge is usually associated with large volume changes of -10 to -30 cm(3) mol(-1), while pressure dependence of K(d)(Zn) resulted in smaller value of DeltaV(d)(Zn), -8.5 cm(3) mol(-1). Therefore, the interaction between ZnCytc and Cytb(5) cannot be explained only by salt bridge interaction, and the partial cancellation by the positive volume change due to the additional hydrophobic interaction is a plausible explanation for the observed DeltaV(d)(Zn). In addition, DeltaV(d)(Zn) of -8.5 cm(3) mol(-1) was considerably smaller than the previously reported volume change, DeltaV(d)(Fe), of -122 cm(3) mol(-1) in the ferric Cytc/Cytb(5) complex dissociation [Rodgers and Sligar (1991) J. Mol. Biol. 221, 1453-1460]. ZnCytc used here has been assumed to be a reliable model of ferrous Cytc, and thus the discrepancy between our present DeltaV(d)(Zn) and the previous DeltaV(d)(Fe) is discussed on the basis of the protein docking dependent on the oxidation states of heme iron in Cytc.

Spectroscopic Characterization of Nonnative Conformational States of Cytochrome c

Protein and heme structural changes of ferric and ferrous cytochrome c (Cyt-c) that are induced by electrostatic binding (e.g., liposomes, electrodes), by hydrophobic interactions (e.g., monomeric sodium dodecyl sulfate), by guanidium hydrochloride (GuHCl), and at low pH and high temperature were studied by UV−vis absorption, circular dichroism (CD), electron paramagnetic resonance (EPR), and (surface-enhanced) resonance Raman [(SE)RR] spectroscopy. In a global spectral analysis, all species that differ with respect to the heme structure were identified and characterized in terms of the spin and ligation state of the heme as well as of protein secondary and tertiary structure changes. The results indicate that the upper part of the heme pocket including the Met-80 ligand is the most labile protein region such that this ligand is dissociated from the heme iron in all nonnative Cyt-c states. Among these states, there are two six-coordinated low-spin (LS) configurations with H2O or His-33 serving as the sixt...

Veratryl alcohol binding sites of lignin peroxidase from Phanerochaete chrysosporium

Possible binding sites of lignin peroxidase (LiP) for veratryl alcohol (VA) were investigated by determining the reactivity of three different chemically modified LiPs against VA when acting (i) as a reducing substrate, (ii) as a rescuing reagent for the rapid conversion of LiPIII ∗ back to native LiP, and (iii) as an enzyme-bound redox mediator. The enzyme was chemically modified to alter its surface properties by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence and the absence of 2-aminoethanesulfonic acid to introduce sulfo and N-acylurea groups instead of carboxylic groups, respectively. LiP was also modified by N-bromosuccinimide (NBS) to yield Trp-modified enzyme. The spectral characteristics and compound I formation rates of modified LiPs were identical to those of unmodified LiP. The activities for VA oxidation by modified LiPs were significantly reduced but with almost unchanged pH dependencies. Several other substrates including phenolic, anionic, and polymeric substrates were also utilized to characterize the activity of the modified enzymes. Kinetic analysis of these reactions strongly suggests that LiP has at least two substrate oxidation sites, one is Trp 171 for VA oxidation and the other is for anionic substrate oxidation. For VA-supported oxidation of ferric cytochrome c (Cc 3+), the VA binding site was estimated to be the same as that of VA oxidation. The reactivities of chemically modified LiPs with excess H 2O 2 were investigated, indicating the formation of compound III ∗ species. Compound III ∗ species of EDC–LiP and unmodified LiP were converted back to ferric enzymes by adding VA. However, there was a reduced recovery of ferric NBS–LiP, suggesting that the VA binding site for VA-derived rapid reversion of compound III ∗ is located at a different site from that for VA oxidation.

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119

Using UV–Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus Solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV–Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo–ferric complex of CYP119, undergoes a ‘peroxide shunt’ pathway during gradual annealing at 170–195 K and returns to the low-spin ferric form.

Mechanism of peroxynitrite interaction with ferric hemoglobin and identification of nitrated tyrosine residues. CO(2) inhibits heme-catalyzed scavenging and isomerization.

Hemoproteins are one of the major targets of peroxynitrite in vivo. It has been proposed that the bimolecular heme/peroxynitrite interaction results in both peroxynitrite inactivation (scavenging) and catalysis of tyrosine nitration. In this study, we used spectroscopic techniques to analyze the reaction of peroxynitrite with human methemoglobin (metHb). Although conventional differential spectroscopy did not reveal heme changes, our results suggest that, in the absence of bicarbonate, the heme in metHb reacts bimolecularly with peroxynitrite but is quickly back-reduced by the reaction products. This hypothesis is based on two indirect observations. First, metHb prevents the peroxynitrite-mediated nitration of a target dipeptide, Ala-Tyr, and second, it promotes the isomerization of peroxynitrite to nitrate. Both the scavenging and the isomerization activities of metHb were heme-dependent and inhibited by CO(2). Ferrous cytochrome c was an efficient scavenger of peroxynitrite, but in the ferric form did not show either scavenging or isomerization activities. We found no evidence of an increase in Ala-Tyr nitration with these hemoproteins. Peroxynitrite-treated metHb induced the formation of a long-lived radical assigned to tyrosine by spin-trapping studies. This radical, however, did not allow us to predict an interaction of peroxynitrite with heme. Hb was nitrated by peroxynitrite/CO(2) mainly in tyrosines beta 130, alpha 42, and alpha 140 and, to a lesser extent, alpha 24. The nitration of alpha chain tyrosines more exposed to the solvent (alpha 140 and alpha 24) was higher in CO-Hb and metHb, while nitration of alpha 42, the tyrosine nearest to the heme, was higher in oxyHb. We deduce that the heme/peroxynitrite interaction, which is inhibited in CO-Hb and metHb, affects alpha tyrosine nitration in two opposite ways, i.e., by protecting exposed residues and by promoting nitration of the residue nearest to the heme. Conversely, nitration of beta Tyr 130 was comparable in oxyHb, metHb, and CO-Hb, suggesting a mechanism involving only nitrating species formed during peroxynitrite decay.