Active Site Of Cytochrome Research Articles

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase

Cytochrome c oxidase (C c O) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a 3 of ba 3 -type C c O. In contrast with the aa 3 -type enzyme, our data show how CO is stabilized on Cu B through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the Cu B -CO complex in ba 3 -type C c O and, by extension, the extremely high oxygen affinity of the enzyme.

A cobalt corrole with a biologically relevant imidazolium pendant for boosted electrocatalytic oxygen reduction

Developing electrocatalysts with high activity and selectivity for the oxygen reduction reaction (ORR) has attracted increasing interest in the past decades. The many imidazole residues located at the active site of cytochrome c oxidases (CcOs) play crucial roles in facilitating the selective reduction of dioxygen to water. As inspired by nature, we herein reported on the synthesis of [Formula: see text] corrole 1 hanged with a biologically relevant imidazolium group and its electrocatalytic ORR features in both acidic and alkaline solutions. Complex 1 is more active and selective than its imidazolium-free analogue for electrocatalytic four-electron ORR. Importantly, 1 displayed ORR activities with the half-wave potentials at [Formula: see text] = 0.65 V versus RHE in 0.5 M [Formula: see text] solution and at [Formula: see text] = 0.81 V versus RHE in 0.1 M KOH solution. This work presents a strategy to simultaneously improve catalytic ORR activity and selectivity by hanging a cationic imidazolium unit over molecular catalytic sites.

Quantification of Local Electric Field Changes at the Active Site of Cytochrome c Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations.

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level.

.Significance: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome oxidase (COX), allowing it to bind instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production.Aim: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells.Approach: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light.Results: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate.Conclusions: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

Non-PGM Electrocatalysts for Oxygen Reduction Reaction Inspired By Metalloenzyme Active Sites

Oxygen reduction reaction (ORR) is a key reaction in future energy generation devices such as polymer electrolyte fuel cells (PEFCs) and metal–air batteries. To drive the ORR in state-of-the-art PEFCs, platinum group metal (PGM) alloys have been used. Since platinum is rare and expensive, there is a need to develop highly active PGM-free ORR electrocatalysts for wide spread applications of PEFCs. In nature, catalytic active sites based on 3d transition metals are widely utilized as catalytic reaction centers in metalloenzymes: for example, laccases and cytochrome c oxidases are known to catalyze the ORR with almost no energy loss at the active sites of multinuclear metal complexes.[1,2] We present ORR electrocatalysts with multinuclear copper active sites incorporated in nitrogen-doped carbon nanosheets, which were inspired by the active site of laccases. Our copper-based electrocatalyst exhibited high ORR activity in alkaline aqueous solution. Physicochemical measurements including in situ X-ray absorption spectroscopy[3] revealed that multinuclear copper sites were embedded in nitrogen-doped carbon nanosheets and worked as the catalytic active center.[4] We also prepared a Fe–Cu-codoped carbon electrocatalyst inspired by the active site of cytochrome c oxidase.[5] Optimization of synthetic conditions and doping ratios of Fe to Cu into carbon resulted in the improvement of the ORR activity in acidic media. The electrocatalyst codoped with Fe and Cu showed higher ORR activity than those with Fe or Cu. Our bioinspired approaches will offer guidance for developing non-PGM ORR electrocatalysts with high activity.

The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase

The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active-site tyrosine (Tyr244) and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Nitrogen Oxide Atom-Transfer Redox Chemistry; Mechanism of NO(g) to Nitrite Conversion Utilizing μ-oxo Heme-Fe(III)-O-Cu(II)(L) Constructs.

While nitric oxide (NO, nitrogen monoxide) is a critically important signaling agent, its cellular concentrations must be tightly controlled, generally through its oxidative conversion to nitrite (NO2(-)) where it is held in reserve to be reconverted as needed. In part, this reaction is mediated by the binuclear heme a3/CuB active site of cytochrome c oxidase. In this report, the oxidation of NO(g) to nitrite is shown to occur efficiently in new synthetic μ-oxo heme-Fe(III)-O-Cu(II)(L) constructs (L being a tridentate or tetradentate pyridyl/alkylamino ligand), and spectroscopic and kinetic investigations provide detailed mechanistic insights. Two new X-ray structures of μ-oxo complexes have been determined and compared to literature analogs. All μ-oxo complexes react with 2 mol equiv NO(g) to give 1:1 mixtures of discrete [(L)Cu(II)(NO2(-))](+) plus ferrous heme-nitrosyl compounds; when the first NO(g) equiv reduces the heme center and itself is oxidized to nitrite, the second equiv of NO(g) traps the ferrous heme thus formed. For one μ-oxo heme-Fe(III)-O-Cu(II)(L) compound, the reaction with NO(g) reveals an intermediate species ("intermediate"), formally a bis-NO adduct, [(NO)(porphyrinate)Fe(II)-(NO2(-))-Cu(II)(L)](+) (λmax = 433 nm), confirmed by cryo-spray ionization mass spectrometry and EPR spectroscopy, along with the observation that cooling a 1:1 mixture of [(L)Cu(II)(NO2(-))](+) and heme-Fe(II)(NO) to -125 °C leads to association and generation of the key 433 nm UV-vis feature. Kinetic-thermodynamic parameters obtained from low-temperature stopped-flow measurements are in excellent agreement with DFT calculations carried out which describe the sequential addition of NO(g) to the μ-oxo complex.

Understanding the mechanism of atovaquone drug resistance in Plasmodium falciparum cytochrome b mutation Y268S using computational methods.

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.

The selective interaction between silica nanoparticles and enzymes from molecular dynamics simulations.

Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the interaction mode between the nano-particles and proteins. In this study, we investigate the orientation and adsorption between several enzymes (cytochrome c, RNase A, lysozyme) and 4 nm/11 nm silica nanoparticles (SNPs) by using molecular dynamics (MD) simulation. Our results show that three enzymes are adsorbed onto the surfaces of both 4 nm and 11 nm SNPs during our MD simulations and the small SNPs induce greater structural stabilization. The active site of cytochrome c is far away from the surface of 4 nm SNPs, while it is adsorbed onto the surface of 11 nm SNPs. We also explore the influences of different groups (-OH, -COOH, -NH2 and CH3) coated onto silica nanoparticles, which show significantly different impacts. Our molecular dynamics results indicate the selective interaction between silicon nanoparticles and enzymes, which is consistent with experimental results. Our study provides useful guides for designing/modifying nanomaterials to interact with proteins for their bio-applications.

CO-dynamics in the active site of cytochrome c oxidase.

The transfer of CO from heme a3 to the Cu(B) site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the Cu(B) site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ≈90% of the ligands are found to rebind to either the Cu(B) (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet.

P63: The antidotal action of nitrites toward cyanide intoxication: An example of medicine disguised as quackery

Sodium nitrite ameliorates sub-lethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. In most experiments nitrite was given intraperitoneally, but may also be administered as an inhaled aqueous vapor-contrary to recommended EMR protocols, it is not necessary to infuse the antidote intravenously and the co-administration of adjuvants (specifically thiosulfate) is unnecessary. Intraperitoneally administered nitrite rapidly produces NO in the bloodstream as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. However, the commonly held belief that the antidotal mechanism of nitrites involves the scavenging of cyanide anion by methemoglobin is firmly contraindicated by recent evidence. Intriguingly, the FDA-approved and clinically tested cyanide scavenger cobalamin has, in fact, rather indifferent binding affinity for cyanide anion. It follows that the clinical usefulness of cobalamin may be dependent on endogenous nitric oxide displacing the cyanide from the active site of cytochrome c oxidase to facilitate the cyanide-scavenging action of cobalamin. Antagonism of cyanide inhibition of cytochrome c oxidase by NO appears to be the crucial antidotal activity rather than any methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood results in the production of sulfidomethemoblobin as detected by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase; the effects of the two inhibitors being essentially additive under standard assay conditions, rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sub-lethal levels of cyanide intoxication. Isoamyl nitrite, given intraperitoneally in equimolar amounts to sodium nitrite, is comparatively less effective at restoring the righting recovery of cyanide-challenged mice. The results of investigations with the hydrolysis products of isoamyl nitrite, isoamyl alcohol and nitrite anion, suggest that the toxicity of isoamyl alcohol renders the organic nitrite a less desirable antidotal agent than sodium nitrite. Disclosure Supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant No. NS063732 to J.P and L.L.P and Bruce R. Pitt.

Heme/Copper Assembly Mediated Nitrite and Nitric Oxide Interconversion

The heme(a3)/Cu(B) active site of cytochrome c oxidase is responsible for cellular nitrite reduction to nitric oxide; the same center can return NO to the nitrite pool via oxidative chemistry. Here, we show that a partially reduced heme/Cu assembly reduces NO(2)(-) ion, producing nitric oxide. The heme serves as the reductant, but the Cu(II) ion is also required. In turn, a μ-oxo heme-Fe(III)-O-Cu(II) complex facilitates NO oxidation to nitrite; the final products are the reduced heme and Cu(II)-nitrito complexes.

Substrate binding and activation in the active site of cytochrome c nitrite reductase: a density functional study

Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His(277), whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg(114). The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr(218), which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.

Reductive Coupling of Nitrogen Monoxide (•NO) Facilitated by Heme/Copper Complexes

The interactions of nitrogen monoxide (*NO; nitric oxide) with transition metal centers continue to be of great interest, in part due to their importance in biochemical processes. Here, we describe *NO((g)) reductive coupling chemistry of possible relevance to that process (i.e., nitric oxide reductase (NOR) biochemistry), which occurs at the heme/Cu active site of cytochrome c oxidases (CcOs). In this report, heme/Cu/*NO((g)) activity is studied using 1:1 ratios of heme and copper complex components, (F(8))Fe (F(8) = tetrakis(2,6-difluorophenyl)porphyrinate(2-)) and [(tmpa)Cu(I)(MeCN)](+) (TMPA = tris(2-pyridylmethyl)amine). The starting point for heme chemistry is the mononitrosyl complex (F(8))Fe(NO) (lambda(max) = 399 (Soret), 541 nm in acetone). Variable-temperature (1)H and (2)H NMR spectra reveal a broad peak at delta = 6.05 ppm (pyrrole) at room temperature (RT), which gives rise to asymmetrically split pyrrole peaks at 9.12 and 8.54 ppm at -80 degrees C. A new heme dinitrosyl species, (F(8))Fe(NO)(2), obtained by bubbling (F(8))Fe(NO) with *NO((g)) at -80 degrees C, could be reversibly formed, as monitored by UV-vis (lambda(max) = 426 (Soret), 538 nm in acetone), EPR (silent), and NMR spectroscopies; that is, the mono-NO complex was regenerated upon warming to RT. (F(8))Fe(NO)(2) reacts with [(tmpa)Cu(I)(MeCN)](+) and 2 equiv of acid to give [(F(8))Fe(III)](+), [(tmpa)Cu(II)(solvent)](2+), and N(2)O((g)), fitting the stoichiometric *NO((g)) reductive coupling reaction: 2*NO((g)) + Fe(II) + Cu(I) + 2H(+) --> N(2)O((g)) + Fe(III) + Cu(II) + H(2)O, equivalent to one enzyme turnover. Control reaction chemistry shows that both iron and copper centers are required for the NOR-type chemistry observed and that, if acid is not present, half the *NO is trapped as a (F(8))Fe(NO) complex, while the remaining nitrogen monoxide undergoes copper complex promoted disproportionation chemistry. As part of this study, [(F(8))Fe(III)]SbF(6) was synthesized and characterized by X-ray crystallography, along with EPR (77 K: g = 5.84 and 6.12 in CH(2)Cl(2) and THF, respectively) and variable-temperature NMR spectroscopies. These structural and physical properties suggest that at RT this complex consists of an admixture of high and intermediate spin states.

Synthesis of a cyclic pentapeptide mimic of the active site His-Tyr cofactor of cytochrome c oxidase.

Arylboronic acid based technology provides a mild, regioselective, and nontoxic N-arylation procedure for accessing the unusual N-arylated side chain histidine found in the active site of cytochrome c oxidase (CcO). The N-arylated histidine is elaborated to the complete cytochrome c oxidase cyclic pentapeptide cofactor. Molecular modeling of the cofactor provides insight into the dynamic character of the N-aryl bond.

Dielectric relaxation of cytochrome c oxidase: Comparison of the microscopic and continuum models

We have studied a charge-insertion process that models the deprotonation of a histidine side chain in the active site of cytochrome c oxidase (CcO) using both the continuum electrostatic calculations and the microscopic simulations. The group of interest is a ligand to Cu(B) center of CcO, which has been previously suggested to play the role of the proton pumping element in the enzyme; the group is located near a large internal water cavity in the protein. Using the nonpolarizable Amber-99 force field in molecular dynamics (MD) simulations, we have calculated the nuclear part of the reaction-field energy of charging of the His group and combined it with the electronic part, which we estimated in terms of the electronic continuum (EC) model, to obtain the total reaction-field energy of charging. The total free energy obtained in this MDEC approach was then compared with that calculated using pure continuum electrostatic model with variable dielectric parameters. The dielectric constant for the "dry" protein and that of the internal water cavity of CcO were determined as those parameters that provide best agreement between the continuum and microscopic MDEC model. The nuclear (MD) polarization alone (without electronic part) of a dry protein was found to correspond to an unphysically low dielectric constant of only about 1.3, whereas the inclusion of electronic polarizability increases the protein dielectric constant to 2.6-2.8. A detailed analysis is presented as to how the protein structure should be selected for the continuum calculations, as well as which probe and atomic radii should be used for cavity definition. The dielectric constant of the internal water cavity was found to be 80 or even higher using "standard" parameters of water probe radius, 1.4 A, and protein atomic radii from the MD force field for cavity description; such high values are ascribed to the fact that the standard procedure produces unphysically small cavities. Using x-ray data for internal water in CcO, we have explored optimization of the parameters and the algorithm of cavity description. For Amber radii, the optimal probe size was found to be 1.25 A; the dielectric of water cavity in this case is in the range of 10-16. The most satisfactory cavity description, however, was achieved with ProtOr atomic radii, while keeping the probe radius to be standard 1.4 A. In this case, the value of cavity dielectric constant was found to be in the range of 3-6. The obtained results are discussed in the context of recent calculations and experimental measurements of dielectric properties of proteins.

Photoinduced Carbon Monoxide Migration in a Synthetic Heme−Copper Complex

Time-resolved infrared (TRIR) flash photolytic techniques have been employed to initiate and observe the efficient dissociation of CO from a synthetic heme-CO/copper complex, [((6)L)Fe(II)(CO)..Cu(I)](+) (2), in CH(3)CN and acetone at room temperature. In CH(3)CN, a significant fraction of the photodissociated CO molecules transiently bind to copper (nu(CO)(Cu) = 2091 cm(-)(1)) giving [((6)L)Fe(II)..Cu(I)(CO)](+) (4), with an observed rate constant, k(1) = 1.5 x 10(5) s(-)(1). That is followed by a slower direct transfer of CO from the copper moiety back to the heme (nu(CO)(Fe) = 1975 cm(-)(1)) with k(2) = 1600 s(-)(1). Additional transient absorption (TA) UV-vis spectroscopic experiments have been performed monitoring the CO-transfer reaction by following the Soret band. Eyring analysis of the temperature-dependent data yields DeltaH(double dagger) = 43.9 kJ mol(-)(1) for the 4-to-2 transformation, similar to that for CO dissociation from [Cu(I)(tmpa)(CO)](+) in CH(3)CN (DeltaH(double dagger) = 43.6 kJ mol(-)(1)), suggesting CO dissociation from copper regulates the binding of small molecules to the heme within [((6)L)Fe(II)..Cu(I)](+)(3). Our observations are analagous to those observed for the heme(a3)/Cu(B) active site of cytochrome c oxidase, where photodissociated CO from the heme(a3) site immediately (ps) transfers to Cu(B) followed by millisecond transfer back to the heme.

Theoretical study of role of H 2O molecule on initial stage of reduction of O 2 molecule in active site of cytochrome c oxidase

Mechanism of formation of [Fe–O–O–H, Cu B] from [Fe–O–O, Cu B] in active site of cytochrome c oxidase was theoretically investigated. It was found that the H 2O molecule is hydrogen-bonded to both Tyr244 and His290, and is a key molecule to produce Fe–O–O–H. This H 2O molecule plays a role as a carrier of a proton toward Fe–O–O to yield [Fe–O–O–H, Cu B]. Cu B atom is formally changed from cuprous state to cupric state through transportation of a proton by the H 2O molecule. A new reaction mechanism, which we call ‘water-proton transport’ (WPT) mechanism, is proposed for the initial stage of reduction of the O 2 molecule.

Nitric Oxide Reduction by Heme‐Thiolate Enzymes (P450nor): A Reevaluation of the Mechanism

AbstractThe details of the heme‐thiolate nitric oxide reductase (P450nor) catalytic mechanism are still controversial. One theory, supported by computational results [D. L. Harris, Int. J. Quantum Chem. 2002, 88, 183−200], assumes two sequential one‐electron transfers from NAD(P)H to an initial [FeNO]6 complex. The [FeNO]8 species thus formed would react with NO, eventually liberating the unstable ONNO2− anion (most probably in its protonated form), which decomposes to N2O and water. However, more recent experimental results [A. Daiber et al., J. Inorg. Biochem. 2002, 88, 343−352] suggest the first committed step of the mechanism to be direct hydride transfer from NAD(P)H to [FeNO]6, presumably resulting in an iron‐bound HNO unit, [Fe‐(H)NO]8, that would be readily protonated to [Fe‐(H)NOH]8. Subsequent NO addition would yield the unstable HO‐N(H)‐N=O, which would dissociate from the heme and decompose to H2O and N2O. Here, the DFT geometry optimization of all previously proposed reaction intermediates is reported. The first step of the mechanism is predicted to be hydride transfer to [FeNO]6, to produce [FeNOH]8 or [Fe‐N(H)O]8. Subsequent addition of NO to [Fe‐NOH]8 (but not to [Fe‐N(H)O]8 or [Fe‐N(H)OH]8) is predicted to lead to immediate liberation of HN2O2−, without any stable intermediates. Contrary to what would be predicted according to the “thiolate push effect” dogma, the thiolate ligand at the heme active site is shown to obstruct NO reduction, rather than facilitate it. It is in fact shown that replacement of the thiolate by a neutral nitrogen ligand (i.e., lysine, as found in the active site of cytochrome c nitrite reductase, an enzyme that can reduce NO) clearly favors, from a thermodynamic point of view, NO reduction at the heme site. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach.

The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochrome b(562), is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochrome b(562) were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000) Biochemistry 39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochrome c' family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cyt b(562) is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 A crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 A resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge-dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (DeltaE(m) = 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.