Heme Environment Research Articles

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch.

It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.

Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

Kinetic properties and heme pocket structure of two domains of the polymeric hemoglobin of Artemia in comparison with the native molecule

In this project, we studied some physicochemical properties of two different globin domains of the polymeric hemoglobin of the brine shrimp Artemia salina and compared them with those of the native molecule. Two domains (AsHbC1D1 and AsHbC1D5) were cloned and expressed in BL21(DE3)pLysS strain of Escherichia coli. The recombinant proteins as well as the native hemoglobin (AfHb) were purified from bacteria and frozen Artemia, respectively by standard chromatographic methods and assessed by SDS-PAGE. The heme environment of these proteins was studied by optical spectroscopy and ligand-binding kinetics (e.g. CO association and O2 binding affinity) were measured for the two recombinant proteins and the native hemoglobin. This indicates that the CO association rate for AsHbC1D1 is higher than that of AsHbC1D5 and AfHb, while the calculated P50 value for AsHbC1D1 is lower than that of AsHbC1D5 and AfHb. The geminate and bimolecular rebinding parameters indicate a significant difference between both domains. Moreover, EPR results showed that the heme pocket in AfHb is in a more closed conformation than the heme pocket in myoglobin. Finally, the reduction potential of −0.13V versus the standard hydrogen electrode was determined for AfHb by direct electrochemical measurements. It is about 0.06V higher than the potential of the single domain AsHbC1D5. This work shows that each domain in the hemoglobin of Artemia has different characteristics of ligand binding.

Activating Biological Functions of Proteins with Gas Molecules

Heme proteins are a class of biological molecules that serve important roles throughout all forms of life. The protein studied in this research, CooA, is found in several bacteria such as Rhodospirillum rubrum (Rr) and Carboxydothermus hydrogenoformans (Ch). In nature, all forms of CooA sense a specific gas molecule, carbon monoxide, that allows the heme protein to bind DNA which activates the transcription process. Interestingly however, in the lab nitric oxide has also been found to activate Ch CooA DNA binding, but it does not activate Rr CooA. It is hypothesized that a specific intermolecular interaction between amino acids within the heme environment is responsible for the different behavior of Ch CooA and Rr CooA. To test this hypothesis, the key amino acids that are proposed to be responsible for this interaction were mutated in both CooA proteins, and the resulting variants were characterized spectroscopically and functionally. The results of this work further develop our understanding of the general gas-sensing mechanism used by heme sensor proteins.

Bacterial Cytochrome c Peroxidases: Insight into the Structure‐Function Relationship

Understanding how a sequence of amino acids serves as a functional, catalytic unit is a fundamental question in enzymology. The Elliott group studies the structure‐function relationship using bacterial cytochrome c peroxidases (bCCPs) as a model system for probing the interplay between structure, redox chemistry, and catalysis. bCCPs are diheme periplasmic enzymes that reduce hydrogen peroxide to water, requiring two c‐type heme cofactors and two electrons from the cytochrome c pool. Peroxide detoxification is an essential bacterial defense mechanism. A hallmark of bCCPs lies in the potentials of their hemes: a low potential peroxidatic heme (FeL) and a high potential electron transfer heme (FeH). Interestingly, the bCCPs from Shewanella oneidensis (SoCCP) and Nitrosomonas europaea (NeCCP) feature a high sequence identity (60%), yet have key catalytic differences: SoCCP requires reductive activation of FeH, where NeCCP does not. Further, the reduction potentials governing respective FeH are very different: where SoCCP (+245 mV vs NHE) is average for bCCPs, and NeCCP (>400 mV vs NHE) is unusually high. Previous experiments in the Elliott group have found that point mutants in one heme environment can globally affect the redox properties. Specifically, mutating a key glutamate to lysine at FeL, which is expected to directly hinder activity, has unexpectedly altered the reduction potentials of FeH as well as FeL. The high sequence and structural similarities, yet differences in electrochemical properties, present a robust model to probe the influence of nearby residues on redox potentials and to understand bacterial defense mechanisms against damaging oxygen species.

Characterization of the Interaction between Heme and a Parallel G-Quadruplex DNA Formed from d(TTAGGGT)

Abstract The molecular structure and heme electronic structure of a complex (heme–(d(TTAGGGT))4 complex) between heme and a parallel G-quadruplex DNA formed from heptanucleotide d(TTAGGGT), i.e., (d(TTAGGGT))4, have been characterized using 1H NMR. The study demonstrated that the heme is accommodated between G6 and T7 of the G-quadruplex DNA to form a 1:1 complex between the heme and DNA. The spin state of the heme Fe3+ of the heme(Fe3+)–(d(TTAGGGT))4 complex exhibited a characteristic pH-dependent change from a high spin (HS) state, i.e., S = 5/2, at low pH to a thermal spin equilibrium between the HS and low spin state (LS), i.e., S = 1/2, at high pH, with a midpoint at the pH value of 8.9 ± 0.2. The pH-dependent spin state change of the heme(Fe3+)–(d(TTAGGGT))4 complex was similar to that of metmyoglobin (T. M. McGrath and G. N. La Mar, Biochim. Biophys. Acta, 1978, 534, 99–111), demonstrating that these two systems are highly alike in terms of the heme environment. This finding provides novel insights as to the design of the heme–DNA complexes that exhibit catalytic activities similar to those of hemoproteins.

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp(309) in substrate binding and catalysis. These studies predicted an elevated pK(a) for Asp(309) and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp(309) with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp(309) based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK(a) of 8.2 observed for androstenedione binding to the side chain of Asp(309). To our knowledge, this work represents the first experimental assignment of a pK(a) value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7-8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.

The Unusual Heme Coordination of THB1, a Hemoglobin from Chlamydomonas Reinhardtii

THB1 is a ∼15 kDa “truncated” hemoglobin (Hb) found in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. As for other Hbs within photosynthetic microbes, THB1 is thought to be involved in the management of reactive oxygen/nitrogen species. To complement physiological studies aimed at determining the function of THB1, we are pursuing the characterization of the recombinant protein (rTHB1) [1] with special attention to the heme environment. At pH ∼ 5, ferric rTHB1 has an optical spectrum characteristic of a six-coordinate, high-spin complex with a histidine and a water molecule as axial ligands to the heme iron. When the pH is raised near neutrality, the protein undergoes a transition (apparent pKa of 6.5) to a six-coordinate, low-spin complex. Mutagenesis and NMR data collected in the diamagnetic ferrous state provide direct evidence for the ligation of a lysine (K53) on the distal side of the heme [1]. Here, we continue the study of this unusual iron coordination scheme. The distal lysine, like the distal histidine of cyanobacterial globins (GlbNs), facilitates electron transfer. However, unlike the histidine of GlbN, the distal lysine of THB1 does not protect ferric heme from oxidative damage caused by H2O2. Analysis of ferrous THB1 NMR data reveals a neutral lysine amino group with highly upfield-shifted 15N signal. Fast-exchange averaging of the two amino protons is a further indication of a dynamic distal coordination. Additional structural information was obtained from diffraction data collected at beamline X25 (National Synchrotron Light Source, Brookhaven National laboratory). Crystals of ferric rTHB1 grown at pH 9.5 diffracted to 1.9 A resolution (P6122 space group) and the data were used to inspect the structural factors allowing lysine coordination.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488

Probing the ligand recognition and discrimination environment of the globin-coupled oxygen sensor protein YddV by FTIR and time-resolved step-scan FTIR spectroscopy.

YddV is a newly discovered signal transducer heme protein that recognizes O2 and CO. Structural differences in the ligand-bound heme complex in YddV reflect variations in catalytic regulation by O2 and CO. Time-resolved step-scan (TRS(2)) FTIR studies of the wild type and of the important in oxygen recognition and stability of the heme Fe(II)-O2 complex L65M, L65T, Y43A, Y43F and Y43W mutants were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand (CO) and also to allow us to investigate the communication pathway between the distal residues of the protein and heme. TRS(2)-FTIR spectra of YddV-heme-CO show that the heme propionates are in protonated and deprotonated states. Moreover, the rate of decay of the vibrations of amide I is on a time scale that coincides with the rate of rebinding of CO, which suggests that there is coupling between ligation dynamics in the distal heme environment and (i) relaxation of the protein backbone and (ii) the environment sensed by the heme propionates. The fast recombination rates in L65M, L65T and Y43W imply a significant role of L65 and Y43 in controlling the ligand dynamics. The implications of these results with respect to the role of the heme propionates and the charged or proton-donating residues in the distal pocket, which are crucial for stabilizing bound gaseous ligands, are discussed.

Heme Coordination Versatility in a Truncated Hemoglobin

GlbN, the “truncated” hemoglobin from Synechocystis sp. PCC 6803, coordinates the heme iron with His46 (E10, distal) and His70 (F8, proximal). Displacement of His E10 by an exogenous ligand (e.g., cyanide in the ferric state) drives a significant conformational change allowing Tyr22 (B10), Gln43 (E7) and Gln47 (E11) to establish a hydrogen bond network stabilizing the distal ligand.THB1, a closely related hemoglobin from Chlamydomonas reinhardtii, also binds cyanide and forms the same network of interactions. However, in the absence of an exogenous ligand, the neutral amino group of Lys53 (E10) coordinates the heme iron on the distal side [1]. Because of the importance of the coordination scheme in controlling the reactivity of heme proteins, the flexibility of the truncated hemoglobin fold, and the rarity of lysine coordination, we explored the ability of GlbN to use lysine at position E10 as an axial ligand. The His46Lys replacement yielded a protein with UV-Vis and NMR spectra similar to those of His46Leu and comparable pH response, suggesting that GlbN does not accommodate lysine coordination at position E10. However, the spectra were incompatible with water coordination and suggested that in the ferric state these variants were low-spin endogenously hexacoordinate complexes. We performed amino acid replacements within the distal H-bond network to characterize the perturbed heme environment and ligand sets. A combination of pH titrations and NMR experiments illustrates the delicate balance of interactions governing the heme pocket conformation of GlbN.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488

A dimeric chlorite dismutase exhibits O2-generating activity and acts as a chlorite antioxidant in Klebsiella pneumoniae MGH 78578.

Chlorite dismutases (Clds) convert chlorite to O2 and Cl–, stabilizing heme in the presence of strong oxidants and forming the O=O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum.

A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625cm(-1), ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'.

Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414nAμM−1) compared to 378nAμM−1 for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74μM) than the HRP biosensor (83μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.

A natural heme-signature variant of CYP267A1 from Sorangiumcellulosum So ce56 executes diverse ω-hydroxylation.

A novel naturally occurring heme-signature variant of CYP267A1 from myxobacterium Sorangiumcellulosum So ce56 and its mutant L366F, the actual mimic of the 'conserved' heme-signature of cytochromes P450, were heterologously expressed in Escherichiacoli in a soluble form and purified. The UV-visible characteristics of both variants were highly similar. Although leucine replaced the phenylalanine in the heme-signature domain of CYP267A1, EPR measurements of the ligand-free wild-type CYP267A1 and the mutant L366F showed low-spin rhombic species suggesting a conserved heme environment of the P450s. The need of primary redox partners for the orphan P450 was sustained by the bovine redox system and a class-I electron transfer path was provided during fatty acid hydroxylation. CYP267A1 showed higher activity and produced more diverse ω-hydroxylated products compared with L366F. In both enzymes the regioselectivity of the fatty acid hydroxylation shifted towards the inner carbon atoms of the fatty acid chains with increasing carbon chain lengths. Our docking results in a homology model of the protein showed that longer fatty acids need to be folded to fit into the binding pocket. In the mutant L366F, the ω-1 and ω-2 positions which exhibit the largest electron density of the highest occupied molecular orbital are preferred. It is speculated that the leucine heme-signature variant of P450 might have evolved under selective evolutionary pressure, which confers an increased advantage to generate a broader spectrum of related alcohols and carboxylic acids required for the bacterial homeostasis or metabolism in a particular ecological niche.

Protein-protein interfaces from cytochrome c oxidase I evolve faster than nonbinding surfaces, yet negative selection is the driving force.

Respiratory complexes are encoded by two genomes (mitochondrial DNA [mtDNA] and nuclear DNA [nDNA]). Although the importance of intergenomic coadaptation is acknowledged, the forces and constraints shaping such coevolution are largely unknown. Previous works using cytochrome c oxidase (COX) as a model enzyme have led to the so-called “optimizing interaction” hypothesis. According to this view, mtDNA-encoded residues close to nDNA-encoded residues evolve faster than the rest of positions, favoring the optimization of protein–protein interfaces. Herein, using evolutionary data in combination with structural information of COX, we show that failing to discern the effects of interaction from other structural and functional effects can lead to deceptive conclusions such as the “optimizing hypothesis.” Once spurious factors have been accounted for, data analysis shows that mtDNA-encoded residues engaged in contacts are, in general, more constrained than their noncontact counterparts. Nevertheless, noncontact residues from the surface of COX I subunit are a remarkable exception, being subjected to an exceptionally high purifying selection that may be related to the maintenance of a suitable heme environment. We also report that mtDNA-encoded residues involved in contacts with other mtDNA-encoded subunits are more constrained than mtDNA-encoded residues interacting with nDNA-encoded polypeptides. This differential behavior cannot be explained on the basis of predicted thermodynamic stability, as interactions between mtDNA-encoded subunits contribute more weakly to the complex stability than those interactions between subunits encoded by different genomes. Therefore, the higher conservation observed among mtDNA-encoded residues involved in intragenome interactions is likely due to factors other than structural stability.

Structures of K42N and K42Y sperm whale myoglobins point to an inhibitory role of distal water in peroxidase activity.

Sperm whale myoglobin (Mb) functions as an oxygen-storage protein, but in the ferric state it possesses a weak peroxidase activity which enables it to carry out H2O2-dependent dehalogenation reactions. Hemoglobin/dehaloperoxidase from Amphitrite ornata (DHP) is a dual-function protein represented by two isoproteins DHP A and DHP B; its peroxidase activity is at least ten times stronger than that of Mb and plays a physiological role. The `DHP A-like' K42Y Mb mutant (K42Y) and the `DHP B-like' K42N mutant (K42N) were engineered in sperm whale Mb to mimic the extended heme environments of DHP A and DHP B, respectively. The peroxidase reaction rates increased ∼3.5-fold and ∼5.5-fold in K42Y and K42N versus Mb, respectively. The crystal structures of the K42Y and K42N mutants revealed that the substitutions at position 42 slightly elongate not only the distances between the distal His55 and the heme iron but also the hydrogen-bonding distances between His55 and the Fe-coordinated water. The enhanced peroxidase activity of K42Y and K42N thus might be attributed in part to the weaker binding of the axial water molecule that competes with hydrogen peroxide for the binding site at the heme in the ferric state. This is likely to be the mechanism by which the relationship `longer distal histidine to Fe distance - better peroxidase activity', which was previously proposed for heme proteins by Matsui et al. (1999) (J. Biol. Chem. 274, 2838-2844), works. Furthermore, positive cooperativity in K42N was observed when its dehaloperoxidase activity was measured as a function of the concentration of the substrate trichlorophenol. This serendipitously engineered cooperativity was rationalized by K42N dimerization through the formation of a dityrosine bond induced by excess H2O2.

Red blood cells polarize green laser light revealing hemoglobin's enhanced non-fundamental Raman modes.

In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measurements. By using malaria diagnosis as an example, we demonstrate that combining the non-fundamental and fundamental regions of the RR spectrum improves the sensitivity and diagnostic capability of the technique. The discovery will have considerable implications for the ongoing development of Raman spectroscopy for blood disease diagnoses and monitoring heme perturbation in response to environmental stimuli.

In vitro and in silico studies of urea-induced denaturation of yeast iso-1-cytochrome c and its deletants at pH 6.0 and 25 °C

Yeast iso-1-cytochrome c (y-cyt-c) has five extra residues at N-terminus in comparison to the horse cytochrome c. These residues are numbered as –5 to –1. Here, these extra residues are sequentially removed from y-cyt-c to establish their role in folding and stability of the protein. We performed urea-induced denaturation of wild-type (WT) y-cyt-c and its deletants. Denaturation was followed by observing change in Δε405 (probe for measuring change in the heme environment within the protein), [θ]405 (probe for measuring the change in Phe82 and Met80 axial bonding), [θ]222 (probe for measuring change in secondary structure) and [θ]416 (probe for measuring change in the heme-methionine environment). The urea-induced reversible denaturation curves were used to estimate Δ, the value of Gibbs free energy change (ΔGD) in the absence of urea; Cm, the midpoint of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Our in vitro results clearly show that except Δ(–5/–4) all deletants are less stable than WT protein. Coincidence of normalized transition curves of all physical properties suggests that unfolding/refolding of WT protein and its deletants is a two-state process. To confirm our in vitro observations, we performed 40 ns MD simulation of both WT y-cyt-c and its deletants. MD simulation results clearly show that extra N-terminal residues play a role in stability but not in folding of the protein.

Regioselective hydroxylation of omeprazole enantiomers by bacterial CYP102A1 mutants.

A large set of Bacillus megaterium CYP102A1 mutants are known to metabolize various drugs to form human metabolites. Omeprazole (OMP), a proton pump inhibitor, has been widely used as an acid inhibitory agent for the treatment of gastric acid hypersecretion disorders. It is primarily metabolized by human CYP2C19 and CYP3A4 to 5'-OH OMP and a sulfone product, respectively. It was recently reported that several CYP102A1 mutants can oxidize racemic and S-OMP to 5'-OH OMP and that these mutants can further oxidize 5'-OH racemic OMP to 5'-COOH OMP. Here, we report that the S- and R-enantiomers of OMP are hydroxylated by 26 mutants of CYP102A1 to produce 1 major metabolite (5'-OH OMP) regardless of the chirality of the parent substrates. Although the binding of R-OMP to the CYP102A1 active site caused a more apparent change of heme environment compared with binding of S-OMP, there was no correlation between the spectral change upon substrate binding and catalytic activity of either enantiomer. The 5'-OH OMP produced from racemic, S-, and R-OMP could be obtained with a high conversion rate and high selectivity when the triple R47L/F87V/L188Q mutant was used. These results suggest that bacterial CYP102A1 mutants can be used to produce the human metabolite 5'-OH OMP from both the S- and R-enantiomers of OMP.

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.