Heme Site Research Articles

Understanding How the Distal Environment Directs Reactivity in Chlorite Dismutase: Spectroscopy and Reactivity of Arg183 Mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.

A Distal Pocket Leu Residue Inhibits the Binding of O2 and NO at the Distal Heme Site of Cytochrome c′

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.

Porphyrins as Detectors of Internal Electric Field in Heme Proteins

Heme proteins display remarkable specificity in discriminating between diatomic ligands that are similar in structure, such as dioxygen, carbon monoxide, and nitric oxide. However, the factors affecting their ability to accomplish this are not known with certainty. We are exploring the influence of the internal electric field produced by the protein matrix on the binding affinities at its heme active site. Generally, the “structure determines function” paradigm is employed to formulate a steric mechanism. Here we present a unique “electrostatic structure” approach to this question, using myoglobin as a model protein. The porphyrin residing at the active site provides a spectroscopic report of the electrostatic environment. In a first step, the atoms comprising the protein matrix are treated as point charges, and their individual electric field contributions are calculated using Coulomb's Law and summed at the heme site. Point mutations surrounding the active site are introduced and the effect on the internal electric field value is noted. In addition, the fluctuation of the field due to natural breathing motions of the protein in solution is modeled, posing the possibility of alternating fields playing a role in ligand discrimination. Experimentally, hole-burning spectroscopy provides a way to extract internal electric field values. This technique uses the Stark effect which is an application of an external electric field perturb the energy of the porphyrin detector. Preliminary results are determined to be quite significant - on the order of megavolts per centimeter. Finally, an excited state analysis of our detector porphyrins using Gaussian09 is presented, including transition dipole moment and state energy values. These values play an important role in the interpretation of spectroscopic data.

Interaction of CORM-2 with hydrophobic sites: Beyond CO

Carbon monoxide releasing molecules (CORMs) have been recently developed for research and pharmacological purposes. A considerable amount of studies demonstrated a wide spectrum of biological activities for lipophilic CORM-2 (tricarbonyldichlororuthenium (II) dimer). It is generally accepted that the liberated gas provides the specific activities to CORMs, with a little attention paid to any possible effect of complementary core molecules. However, the versatile repertoire of actions attributed to CORM-2 is surprisingly wide for CO, a molecule with the sole chemical activity of binding to ferrous iron in protein prosthetic groups. The study was designed to analyze CORM-2 and its core molecule (“i”CORM) activities at a molecular level. With respect to the hydrophobic nature of the compounds, we followed their interactions with several amphipathic entities: the heme sites of hemoproteins, heme binding proteins and cell membranes. CORM-2/“i”CORM decreased the Soret optical density of hemoglobin and myoglobin, indicating that both compounds interact with the protein amphipathic site in the heme pocket. Pre-addition of CORM-2/“i”CORM to the apo-forms of the plasma heme binding proteins, hemopexin and albumin, partially abolished their heme binding capacity. In contrast, the compounds had no effect on the preformed heme-protein complexes. Addition of CORM-2/“i”- CORM to blood or isolated erythrocytes revealed aggregation of the cells or lysis, depending on the rea-gent-to-cells ratio. It was concluded that the ruthenium containing core molecule of CORM-2 may be physiologically active due to non-specific hydrophobic interactions. As each type of CORMs is expected to have a different mode of action beyond CO activity, their potential therapeutic uses will require clarification.

Direct Observation of Vibrational Energy Flow in Cytochrome c

Vibrational energy flow in ferric cytochrome c has been examined by picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) measurements. By taking advantage of the extremely short nonradiative excited state lifetime of heme in the protein (<< ps), excess vibrational energy of 20000-25000 cm(-1) was optically deposited selectively at the heme site. Subsequent energy relaxation in the protein moiety was investigated by monitoring the anti-Stokes UVRR intensities of the Trp59 residue, which is a single tryptophan residue involved in the protein that is located close to the heme group. It was found from temporal changes of the anti-Stokes UVRR intensities that the energy flow from the heme to Trp59 and the energy release from Trp59 took place with the time constants of 1-3 and ~8 ps, respectively. These data are consistent with the time constants for the vibrational relaxation of the heme and heating of water reported for hemeproteins. The kinetics of the energy flow were not affected by the amount of excess energy deposited at the heme group. These results demonstrate that the present technique is a powerful tool for studying the vibrational energy flow in proteins.

Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c-NO and 6c-CO complexes but do not form O(2) complexes. Structure of the AxCYTcp-CO complex at 1.25 Å resolution shows that CO binds in two conformations moderated by the extent of displacement of the distal residue Leu16 toward the haem 7-propionate. The presence of two CO conformations is confirmed by cryogenic resonance Raman data. The preferred linear Fe-C-O arrangement (170 ± 8°) is accompanied by a flip of the propionate from the distal to proximal face of the haem. In the second conformation, the Fe-C-O unit is bent (158 ± 8°) with no flip of propionate. The energetic cost of the CO-induced Leu-propionate movements is reflected in a 600 mV (57.9 kJ mol(-1)) decrease in haem potential, a value in good agreement with density functional theory calculations. Substitution of Leu by Ala or Gly (structures determined at 1.03 and 1.04 Å resolutions) resulted in a haem site that binds CO in the linear mode only and where no significant change in redox potential is observed. Remarkably, these variants were isolated as ferrous 6c-CO complexes, attributable to the observed eight orders of magnitude increase in affinity for CO, including an approximately 10,000-fold decrease in the rate of dissociation. These new findings have wide implications for preventing CO poisoning of gas-binding haem proteins.

Ligand Binding Intermediates of Nitrosylated Human Hemoglobin Induced at Low Temperature by X-ray Irradiation

Under prolonged X-ray irradiation, the ferrous heme of nitrosylated human adult hemoglobin derivative (HbNO) undergoes a reversible transition generating a 5-coordinate species, due to release of the Fe-NO bond. The overall process can be investigated using X-ray absorption near edge structure (XANES) spectroscopy. In this work, Fe K-edge XANES spectra were measured at T < 15 K, pH 9.2, i.e., on a high-affinity state (R-HbNO) where all the hemes are 6-coordinate, and at pH 6.5 in the presence of inositol hexakis-phosphate (IHP), i.e., on a low-affinity ligated state (T-HbNO) where the iron-hemes of the α-chains are 5-coordinate due to breaking of the Fe-proximal histidine bond. Under X-ray irradiation, 5-coordinate Fe-hemes are populated in both R-HbNO and T-HbNO, the Fe-NO bond lysis induced in T-HbNO involving rebinding of the proximal histidine to the transiently populated 4-coordinate hemes of the α-chains. A detailed analysis of the spectra confirms that different intermediate states in the ligand binding cooperative process of hemoglobin can be populated by X-ray irradiation, and that the part of the energy associated to the R-T quaternary transition, that is transmitted to the heme site, can be monitored by XANES spectroscopy.

Insights into Structure and Function of the Active Site of SoxAX Cytochromes

SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met(236), representing an unusual rotamer of methionine. The flexibility of the SnSoxAX(C236M) SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, -479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s(-1); C236M, 2.0 s(-1)). SnSoxAX(C236M) was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys(236) aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.

Differential effects of glutamate-286 mutations in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Both the aa 3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsC cO aa3 ) and the closely related bo 3-type ubiquinol oxidase from Escherichia coli (EcQO bo3 ) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsC cO aa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a 3 . Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsC cO aa3 to ~ 0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a 3-Cu B active site. In contrast, the E286C mutation of EcQO bo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsC cO aa3 , the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO bo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Predicting the functionally distinct residues in the heme, cation, and substrate-binding sites of peroxidase from stress-tolerant mangrove specie, Avicennia marina

Recent work was conducted to predict the structure of functionally distinct regions of Avicennia marina peroxidase (AP) by using the structural coordinates of barley grains peroxidase as the template. This enzyme is utilized by all living organisms in many biosynthetic or degradable processes and in defense against oxidative stress. The homology model showed some distinct structural changes in the heme, calcium, and substrate-binding regions. Val53 was found to be an important coordinating residue between distal calcium ion and the distal heme site while Ser176 is coordinated to the proximal histidine through Ala174 and Leu172. Different ionic and hydrogen-bonded interactions were also observed in AP. Analyses of various substrate-enzyme interactions revealed that the substrate-binding pocket is provided by the residues, His41, Phe70, Gly71, Asp138, His139, and Lys176; the later three residues are not conserved in the peroxidase family. We have also performed structural comparison of the A. marina peroxidase with that of two class III salt-sensitive species, peanut and soybean. Four loop regions were found to have largest structural deviation. The overall protein sequence was also analyzed for the presence of probable post-translational modification sites and the functional significance of these sites were outlined.

Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization.

• Irr binds to irr by molecular sieving (View Interaction 1, 2) • Irr binds to irr by cosedimentation in solution (View interaction).

Novel flavohemoglobins of mycobacteria

Flavohemoglobins (flavoHbs) constitute a distinct class of chimeric hemoglobins in which a globin domain is coupled with a ferredoxin reductase such as FAD- and NADH-binding modules. Structural features and active site of heme and reductase domains are highly conserved in various flavoHbs. A new class of flavoHbs, displaying crucial differences in functionally conserved regions of heme and reductase domains, have been identified in mycobacteria. Mining of microbial genome data indicated that the occurrence of such flavoHbs might be restricted to a small group of microbes unlike conventional flavoHbs that are widespread among prokaryotes and lower eukaryotes. One of the representative flavoHbs of this class, encoded by Rv0385 gene (MtbFHb) of Mycobacterium tuberculosis, has been cloned, expressed, and characterized. The ferric and deoxy spectra of MtbFHb displayed a hexacoordinate state indicating that its distal site may be occupied by an intrinsic amino acid or an external ligand and it may not be involved in nitric oxide detoxification. Phylogenetic analysis revealed that mycobacterial flavoHbs constitute a separate cluster distinct from conventional flavoHbs and may have novel function(s).

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe³(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.

Dynamics Investigation of the Cytochrome P450cam Active Site Mutant Thr252ALA

Cytochrome P450s are vital heme-containing enzymes that facilitate catalysis in a wide variety of biochemical processes including carcinogenesis, drug metabolism, steroid biosynthesis and toxin degradation. Because of its ability to catalyze the hydroxylation mechanism of camphor in its active site, Cytochrome P450cam served as the model system for studying P450s. The crystal structure of a P450cam mutant, in which the active site Threonine252 (Thr252) has been replaced with an alanine (ala) residue (Thr252Ala), was solved at 2.2Å resolution. According to sequence alignment analysis, Thr252 is highly conserved among P450s, and it was suggested to be an essential active site residue, forming part of the dioxygen-binding site. Mutation of the active site Thr to Ala changes local conformation of the active site residues and affects hydroxylation of the substrate, producing hydrogen peroxide and “excess” water instead of the substrate polarizing product, 5-exo-hydroxycamphor. In the mutant active site, a solvent not present in the native enzyme is positioned in the dioxygen-binding region and the network of hydrogen bonds is restructured accordingly. These factors are believed to be critical to enzymatic reaction changes of P450cam mutant from the wild-type, most notably the proton delivery. Molecular dynamics (MD) simulation has been performed on the mutant to predict the relevant structural dynamics, especially those of the active site residues. Neutron scattering (NS) spectra calculations have been extrapolated from the simulation and compared with the results with the indicated crystallographic findings and those of the wild-type P450cam simulations already empirically verified via experimental neutron scattering. Preliminary results predict that local interactions around the heme and cam site have been altered midway through the trajectory. The preliminary data has also provided insight to understanding the local water diffusion dynamics of the CYP450cam mutant active site.

Axial ligation of the high-potential heme center in an Arabidopsis cytochrome b561

Arabidopsis has four putative, di-heme cytochrome b561 proteins, including one localized in the tonoplast (TCytb). From a comparative electron paramagnetic resonance (EPR), UV–Vis absorption and resonance Raman study, on wild type, H83A/H156A-TCytb and H83L/H156L-TCytb double mutants, it follows that the H83 and H156 residues are binding one of the two hemes. These measurements show that the high-potential heme site is situated at the cytoplasmic side of the membrane and allow the unambiguous differentiation between two models on the heme localization in cytochrome b561 proteins.

Crystal Structure of HugZ, a Novel Heme Oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.

X-Ray-Induced Lysis of the Fe−CO Bond in Carbonmonoxy-Myoglobin

By using X-ray absorption near edge structure (XANES) spectroscopy, we show that under prolonged exposure to Synchrotron X-rays, at T < 10 K, the Fe-heme in carbonmonoxy-myoglobin (MbCO) undergoes a slow two-state transition process. The final spectrum is nearly identical to that of the classical photoproduct (Mb*CO) obtained by UV-visible light illumination at 15 K. By increasing the temperature, the starting spectrum of MbCO is recovered at T > 100 K, demonstrating that the process is reversible and no damage occurred at the heme site in the time course of the experiment. Thus, the overall X-ray-induced process at low temperature is identical to the well-known (light-induced) photolysis of CO-hemeproteins.

Redox regulation of guanylate cyclase and protein kinase G in vascular responses to hypoxia.

The production of cGMP by the soluble form of guanylate cyclase (sGC) in bovine pulmonary arteries (BPA) is controlled by cytosolic NADPH maintaining reduced thiol and heme sites on sGC needed for activation by NO, and the levels of Nox oxidase-derived superoxide and peroxide that influence pathways regulating sGC activity. Our recent studies in BPA suggest that the activities of peroxide metabolizing pathways in vascular smooth muscle potentially determine the balance between sGC stimulation by peroxide and a cGMP-independent activation of cGMP-dependent protein kinase (PKG) by a disulfide-mediated subunit dimerization. Cytosolic NADPH oxidation also appears to function in BPA through its influence on protein thiol redox control as an additional mechanism promoting vascular relaxation through PKG activation. These processes regulating PKG may participate in decreases in peroxide and increases in NADPH associated with contraction of BPA to hypoxia and in cytosolic NADPH oxidation potentially mediating bovine coronary artery relaxation to hypoxia.

Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Mammalian nitric oxide synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide (NO). Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the flavin mononucleotide (FMN) to heme domains is essential in the delivery of electrons required for O(2) activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe...FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH(*)] (FMNH(*): FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH(*)...Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 +/- 0.1 A) is in excellent agreement with the IET rate constant measured by laser flash photolysis [Feng, C. J.; Dupont, A.; Nahm, N.; Spratt, D.; Hazzard, J. T.; Weinberg, J.; Guillemette, J.; Tollin, G.; Ghosh, D. K. J. Biol. Inorg. Chem. 2009, 14, 133-142].

Mechanisms of Peroxynitrite Interactions with Heme Proteins

Oxygenated heme proteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. PN is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN interactions with heme proteins. In this Forum, we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve NO and PN. We have recently described direct evidence that ferrylmyolgobin (ferrylMb) and nitrogen dioxide (NO(2)) are both produced during the reaction of PN and metmyolgobin (metMb) (Su, J.; Groves, J. T. J. Am. Chem. Soc. 2009, 131, 12979-12988). Kinetic evidence indicates that these products evolve from the initial formation of a caged radical intermediate [Fe(IV) horizontal lineO.NO(2)]. This caged pair reacts mainly via internal return with a rate constant k(r) to form metMb and nitrate in an oxygen-rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, k(obs), of heme-mediated PN decomposition. Freely diffusing NO(2), which is liberated concomitantly from the radical pair (k(e)), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a beta-sheet structure, at the expense of turns and helices at low pH (Balakrishnan, G.; Hu, Y.; Oyerinde, O. F.; Su, J.; Groves, J. T.; Spiro, T. G. J. Am. Chem. Soc., 2007, 129, 504-505). It is proposed that a short beta-sheet segment, comprising residues 37-39 and 58-61, extends itself into the large 37-61 loop when the latter is destabilized by protonation of H26, which forms an anchoring hydrogen bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in ligation-sensitive Raman bands. It also induces peroxidase activity with the same temperature profile. This process is suggested to model the apoptotic peroxidation of cardiolipin by cytochrome c.