Heme Active Site Research Articles

Structural and functional alterations of myoglobin by glucose-protein interactions

The interaction of blood glucose with heme proteins plays a key role in inducing diabetes, a serious disease threatening human health. In this study, we investigated the non-covalent interaction between glucose and myoglobin (Mb), both theoretically and experimentally, using molecular dynamics (MD) simulation combined with spectroscopic studies. It revealed that glucoses can occupy the side pocket of Mb, and bind closely to one of the xenon cavities in Mb, by hydrogen bonding interactions with two propionate groups of heme as well as surrounding amino acids. These interactions alter the conformation of the heme active site slightly and lead to an enhanced peroxidase activity of Mb, as determined by kinetic studies. This study provides general information for glucose-heme proteins interactions, and also for blood glucose-protein interactions for patients with diabetes.

Understanding how the distal pocket environment affects the ligand binding affinity of nitrite to heme proteins (1013.17)

Cardiovascular disease is the leading cause of death worldwide. Nitric oxide (NO) plays a fundamental role in cardiovascular health and disruptions in normal NO physiology is associated with the progression of cardiovascular disease. A relatively new mechanism by which heme proteins can support vasodilatation during hypoxia is by converting nitrite (NO2‐) to NO. This NO2‐ reductase activity plays essential roles in a variety of physiological processes. Because there is a large range in NO2‐ affinity and NO2‐ reductase activity in heme proteins with the same active site, this research seeks to better understand how the protein environment controls the binding chemistry of NO2‐ to the heme active site. Our goal is to determine how the distal pocket environment affects the binding affinity of NO2‐ to metmyoglobin mutants. Our hypothesis is that the distal pocket environment can increase the binding affinity of NO2‐ by way of electrostatic and steric interactions. To do this, we will compare the binding affinity of a series of distal pocket mutants that affect hydrogen bonding, polarity, and the size of the distal pocket. We will correlate coordination chemistry and binding affinity in order to establish a clear picture of how the protein environment controls NO2‐ binding affinity in heme proteins. We found that polar distal residues exhibit greater binding affinity to NO2‐ than non‐polar residues due to hydrogen bonding. These results indicate that proteins with polar distal residues will exhibit greater NO2‐ reductase activity. Understanding how the protein environment influences NO2‐ binding in heme proteins helps toward understanding how these proteins generate NO physiologically, and in designing therapeutics based on the NO2‐ reductase activity of heme proteins.

Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs

Myeloperoxidase (MPO) catalyzes the breakdown of hydrogen peroxide and the formation of the potent oxidant hypochlorous acid. We present the application of the fluorogenic peroxidase substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in steady-state and transient kinetic studies of MPO function. Using initial kinetic parameters for the MPO system, we characterized under the same conditions a number of gold standards for MPO inhibition, namely 4-amino benzoic acid hydrazide (4-ABAH), isoniazid and NaN3 before expanding our focus to isomers of 4-ABAH and benzoic acid hydrazide analogs. We determined that in the presence of hydrogen peroxide that 4-ABAH and its isomer 2-ABAH are both slow-tight binding inhibitors of MPO requiring at least two steps, whereas NaN3 and isoniazid-based inhibition has a single observable step. We also determined that MPO inhibition by benzoic acid hydrazide and 4-(trifluoromethyl) benzoic acid hydrazide was due to hydrolysis of the ester bond between MPO heavy chain Glu 242 residue and the heme pyrrole A ring, freeing the light chain and heme b fragment from the larger remaining MPO heavy chain. This new mechanism would essentially indicate that the benzoic acid hydrazide analogs impart inhibition through initial ejection of the heme catalytic moiety without prior loss of the active site iron.

Binding of the Antitubercular Pro-Drug Isoniazid in the Heme Access Channel of Catalase-Peroxidase (KatG). A Combined Structural and Metadynamics Investigation

Isonicotinic acid hydrazide (isoniazid or INH) is a front line antitubercular pro-drug that is converted to its active form, isonicotinyl-NAD, by the bacterial catalase-peroxidase KatG. Understanding the role of KatG in the INH activation process has been hampered by a lack of knowledge of the actual drug binding site. In this work, we have investigated the binding of INH in the main access channel of KatG with a combination of molecular dynamics, using an enhanced-sampling technique (metadynamics), X-ray crystallography, and site-directed mutagenesis. The metadynamics simulations show that there are several weak drug binding sites along the access channel. Moreover, the simulations evidence that complete entrance to the heme active site is impeded by an aspartate residue (D141) located above the heme. This has been confirmed by structural and functional analysis of the D141A mutant, leading to the first X-ray crystallography evidence of INH at the heme access channel.

Co Recombination in Human IDO and TDO - A Comparison

The heme enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring and the insertion of molecular oxygen to produce N-formylkynurenine, the first and rate-limiting step in the kynurenine pathway.The catalytic reaction involves the formation of a ternary complex including the protein, the ligand (O2), which binds to the ferrous heme iron, and the substrate (L-Trp). For optimal turnover of the enzyme, the protein scaffold has to provide easy access for both O2 and L-Trp to the heme active site. Efficient O2 association may require transient docking sites to be accessible. As both oxygen atoms are inserted into the C2 - C3 bond of the L-Trp pyrrole ring during the reaction, efficient ligand escape is (most likely) not required. L-Trp binding calls for a rather large 'access gate'. In addition, the reaction product N-formylkynurenine must be expelled from the active site to allow the uptake of new reagents.The molecular details of the catalytic mechanisms employed by TDO and IDO have, as yet, not been elucidated. Therefore, we have investigated ligand rebinding in ferrous IDO and TDO using flash photolysis at ambient temperatures, replacing the physiological ligand O2 by CO, to disclose both similarities of and differences between the enzymes. CO allows us to exploit its excellent properties as a spectroscopic probe. Moreover, CO is not reactive towards L-Trp, so that we can focus on ligand and substrate dynamics in the absence of the ensuing enzymatic reaction.

Reduction of the internal disulfide bond between Cys 38 and 83 switches the ligand migration pathway in cytoglobin

Despite the similar tertiary structure between cytoglobin (Cygb) and myoglobin, several structural features indicate a distinct mechanism of Cygb interactions with exogenous ligands. Here we present a spectroscopic investigation of the dynamics and thermodynamics of structural changes associated with the exogenous ligand migration between the solvent and the heme active site in Cygb with reduced and oxidized Cys 38 and Cys 83 side-chains (Cygbox and Cygbred, respectively). Photo-acoustic and transient absorption data show that disulfide bond formation alters the ligand migration pathway(s) as evident from the distinct geminate quantum yields (Φgem=0.35 for Cygbox and Φgem=0.63 for Cygbred) and rate constants for bimolecular CO rebinding. Moreover, ligand escape from the protein matrix is fast (<40ns) and coupled with an enthalpy change of 18±2kcalmol−1 in Cygbred, whereas the disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4kcalmol−1. These results demonstrate that the disulfide bond connecting helix E and helix B modulates the conformational dynamics in Cygb including the size and energy barrier between the internal hydrophobic sites. Based on the comparison of the thermodynamic profiles for CO photo-dissociation from Cygb, myoglobin, and neuroglobin we propose that in Cygbred the photo-dissociated ligand escapes through the hydrophobic tunnel, whereas the CO preferably migrates through the His64 gate in Cygbox suggesting that Cygb's physiological role may vary in response to intracellular redox conditions.

Structure of the processive rubber oxygenase RoxA from Xanthomonas sp

Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 Å. The enzyme is a 75-kDa diheme c-type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O2 stably bound to the active site heme iron. Activation and cleavage of O2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.

Engineering of a hybrid biotransformation system for cytochrome P450sca‐2 inEscherichia coli

P450sca-2 is an industrially important enzyme that stereoselectively converts mevastatin into pravastatin. However, little information or engineering efforts have been reported for this enzyme or its redox partner. In this study, we successfully reconstituted the P450sca-2 activity in Escherichia coli by co-expression with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from the Pseudomonas putida cytochrome P450cam system. With an HPLC-based screening assay, random mutagenesis was applied to yield a mutant (R8-5C) with a pravastatin yield of the whole-cell biotransformation 4.1-fold that of the wild type. P450sca-2 wild-type and R8-5C were characterized in terms of mevastatin binding and hydroxylation, electron transfer, and circular dichroism spectroscopy. R8-5C showed an active P450 expression level that was 3.8-fold that of the wild type, with relatively smaller changes in the apparent k(cat)/K(M) with respect to the substrate mevastatin (1.3-fold) or Pdx (1.5-fold) compared with the wild type. Thus, the increase in the pravastatin yield of the whole-cell biotransformation primarily came from the improved active P450 expression, which has resulted largely from better heme incorporation, although none of the six mutations of R8-5C are located near the heme active site. These results will facilitate further engineering of this P450sca-2 system and provide useful clues for improving other hybrid P450 systems.

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a≈7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current-potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

Distinct Structural and Redox Properties of the Heme Active Site in Bacterial Dye Decolorizing Peroxidase-Type Peroxidases from Two Subfamilies: Resonance Raman and Electrochemical Study

Spectroscopic data of dye decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP), an A subfamily member, and Pseudomonas putida (PpDyP), a B subfamily enzyme, reveal distinct heme coordination patterns of the respective active sites. In solution, both enzymes show a heterogeneous spin population, with the six-coordinated low-spin state being the most populated in the former and the five-coordinated quantum mechanically mixed-spin state in the latter. We ascribe the poor catalytic activity of BsDyP to the presence of a catalytically incompetent six-coordinated low-spin population. The spin populations of the two DyPs are sensitively dependent on the pH, temperature, and physical, i.e., solution versus crystal versus immobilized, state of the enzymes. We observe a redox potential for the Fe(2+)/Fe(3+) couple in BsDyP (-40 mV) at pH 7.6 substantially more positive than those reported for the majority of other peroxidases, including PpDyP (-260 mV). Furthermore, we evaluate the potential of the studied enzymes for biotechnological applications on the basis of electrochemical and spectroelectrochemical data.

Structural and nitrite reductase activity comparisons of myoglobins with one to three distal histidines

Although with a distinct heme active site, both myoglobin (Mb) and cytochrome c oxidase (CcO) were found to function as a nitrite reductase (NIR) under hypoxic conditions. On the other hand, Mb was rationally designed to mimic native CcO by introduction of two distal histidines, i.e., L29H/F43H mutant, where His29, His43 and native His64 formed a metal-binding site. To probe the role of distal histidines in regulating the NIR activity of Mb, we herein designed a single mutant of L29H Mb that contains two distal histidines and solved its X-ray crystal structure, then we made both structural and NIR activity comparisons of Mbs with one to three distal histidines. It was shown that introduction of one or two additional distal histidines in Mb leads to the formation of a distal hydrogen network, which slightly alters the conformation of heme active site and inhibits the NIR activity. Meanwhile, the reductase activity of both L29H Mb and L29H/F43H Mb are regulated by metal ions such as Cu(II) or Zn(II) binding to the distal histidines, i.e., Cu(II) enhances the reactivity while Zn(II) inhibits the reactivity. These findings provide valuable insights into the structure and function relationship for both Mb and CcO functioning as a NIR in biological system.

Comparison of the Crystal and Solution Structures of a Cyanobacterial Hemoglobin

GlbN is a monomeric 2/2 hemoglobin found in several cyanobacteria. Physiological data suggest that, in Synechococcus sp. PCC 7002, GlbN protects the cell from damage associated with nitrate metabolism [1]. Under reducing conditions, GlbN undergoes a facile posttranslational cross-linking between His117 and the heme 2-vinyl. In a study of the enzymatic and chemical properties of GlbN, the solution structure of the protein in the ferric, bis-histidine state with heme posttranslational modification was determined by NMR methods. As this protein is paramagnetic (S = 1/2) and can be prepared in the reduced state (S = 0), heme methyl hyperfine chemical shifts were used to determine the orientation of the ligating histidines, and proton pseudocontact shifts were applied in the refinement. The NMR model contains regions that appear to sample multiple conformations, specifically the A helix and EF loop. Here we present the X-ray structure of GlbN. Crystals were obtained by the vapor diffusion hanging drop method, and data were collected under cryogenic conditions (National Synchrotron Light Source, BNL). The structure of the related Synechocystis sp. PCC 6803 GlbN (1RTX) was used as the search model for molecular replacement. Iterative model building yielded a final structure at 1.65 A resolution. The X-ray data revealed a ruffled heme, provided a reliable representation of its environment in solution, and allowed for an assessment of the paramagnetic NMR approach. The X-ray data also gave an indication of the effect of crystal packing on the flexible regions of GlbN. Comparison with the Synechocystis GlbN structure highlighted the resilience of the 2/2 globin fold as well as subtle differences in the solvent accessibility of the heme active site.[1] Scott et al. (2010) Biochemistry 49:7000Supported by NSF MCB 0843439

Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). An active site heme ligand becomes labile on exposure to substrate analogs. Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis.

Functional Importance of a Pair of Conserved Glutamic Acid Residues and of Ca2+ Binding in the cbb3-Type Oxygen Reductases from Rhodobacter sphaeroides and Vibrio cholerae

The cbb(3)-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb(3)-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two b-type hemes, three c-type hemes, and Cu(B)), the enzyme also contains at least one Ca(2+). The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b(3) and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca(2+) is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In this work, mutations of these glutamates are investigated in the cbb(3)-type oxidases from Vibrio cholerae and Rhodobacter sphaeroides. Metal analysis shows that each of these wild-type enzymes contains Ca(2+). Mutations of the glutamate expected to ligate the Ca(2+) in each of these enzymes (E126 in V. cholerae and E180 in R. sphaeroides) result in a loss of activity as well as a loss of Ca(2+). Mutations of the nearby glutamate (E129 in V. cholerae and E183 in R. sphaeroides) also resulted in a loss of oxidase activity and a loss of Ca(2+). It is concluded that the Ca(2+) is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb(3)-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.

The bacterial SoxAX cytochromes.

SoxAX cytochromes are heme-thiolate proteins that play a key role in bacterial thiosulfate oxidation, where they initiate the reaction cycle of a multi-enzyme complex by catalyzing the attachment of sulfur substrates such as thiosulfate to a conserved cysteine present in a carrier protein. SoxAX proteins have a wide phylogenetic distribution and form a family with at least three distinct types of SoxAX protein. The types of SoxAX cytochromes differ in terms of the number of heme groups present in the proteins (there are diheme and triheme versions) as well as in their subunit structure. While two of the SoxAX protein types are heterodimers, the third group contains an additional subunit, SoxK, that stabilizes the complex of the SoxA and SoxX proteins. Crystal structures are available for representatives of the two heterodimeric SoxAX protein types and both of these have shown that the cysteine ligand to the SoxA active site heme carries a modification to a cysteine persulfide that implicates this ligand in catalysis. EPR studies of SoxAX proteins have also revealed a high complexity of heme dependent signals associated with this active site heme; however, the exact mechanism of catalysis is still unclear at present, as is the exact number and types of redox centres involved in the reaction.

Catalases versus peroxidases: DFT investigation of H2O2 oxidation in models systems and implications for heme protein engineering

Catalases and peroxidases are ubiquitous heme enzymes that catalyze the removal of hydrogen peroxide (H2O2). Both enzymes use one molecule of hydrogen peroxide to form a high valent iron intermediate named Compound I (Cpd I). However, whereas catalase Cpd I oxidizes a second H2O2 molecule to oxygen, peroxidases use this intermediate to oxidize other substrates rather than H2O2. The origin of the different reactivity of peroxidases and catalases is not known, but it is likely to be related to structural differences between the two heme active sites. Recent modeling studies suggest that the oxidation of H2O2 by catalase Cpd I may take place by two hydrogen atom transfer steps. In this work, we investigate how catalases and peroxidases compare along the same hydrogen transfer steps to give hints into the question why peroxidases cannot efficiently oxidize H2O2. The use of simplified models allows us to probe the direct effect of the proximal ligand (tyrosinate in catalases and histidine in peroxidases) without masking from the protein environment. We show that the nature of the fifth ligand (His in peroxidase and Tyr in catalase) has little effect on the energy barriers of the hydrogen transfer steps. On the contrary, the Cpd I–hydrogen peroxide (OFe–Operoxide) distance affects significantly the reaction barriers. We propose that the distal side architecture of peroxidases do not allow to attain short OCpd I–Operoxide distances, thus resulting in a lower efficiency towards H2O2 oxidation.

Role of Hsp90- and Hsp70-chaperones in regulation of neuronal NO-synthase ubiquitination and proteasomal degradation

The metabolism-based inactivation of neuronal NO-synthase (nNOS) leads to the covalent alteration of the nNOS heme active site and proteasomal degradation of the inactivated nNOS. The mechanism of how the inactivated nNOS is selectively culled for degradation is not known. We have previously shown that nNOS turnover is regulated by Hsp70/CHIP (C-terminus of Hsp70-interacting protein)-dependent ubiquitination. We now show that xenobiotic-inactivated nNOS is selectively ubiquitinated by CHIP in an in vitro system containing purified proteins. Hsp70 facilitates the ubiquitination while Hsp90 protects nNOS from ubiquitination. A C331A mutant of nNOS, which was previously characterized by Dr. Masters’s lab as having an altered active site conformation, is labilized for Hsp70/CHIP-dependent ubiquitination. The slowly reversible inhibitor, NG-nitro- l -arginine (NNA), but not the D-isomer of NNA, protects the C331A and wild type nNOS from ubiquitination in in vitro systems as well as in intact cells. More recently, we found that NNA decreases binding of Hsp70 and CHIP to nNOS. Studies where the oxygenase and reductase domains of nNOS were expressed in HEK293 cells show that Hsp70 and Hsp90 recognize the oxygenase domain. Thus, conformational changes about the heme active site appear to be recognized by chaperones, which then direct the CHIP-dependent ubiquitination. In that clefts are important topological features in nearly all proteins, the ability to detect these conformational changes maybe a mechanism for maintaining nNOS protein quality through chaperones, as well as play an important role in the chaperone-facilitated insertion of heme into the active site cleft of apo-nNOS (Supported by NIH GM077430).

Nitric oxide synthases in motion

To determine a dynamic functional model of full-length nitric oxide synthase (NOS), we are coupling Small Angle X-ray Scattering (SAXS) with our crystal structures of individual NOS components. SAXS is a solution scattering method that is well suited to capturing protein conformational changes. Our lab has solved crystal structures of all three parts of NOS enzymes: NOS oxygenase, NOS reductase and NOS calmodulin (CaM) peptide with CaM bound. Based on our crystal structures and biochemical data, we propose that the NOS FMN domain swings from the electron-accepting “IN” to an electron-donating “OUT” conformation. In the electron-accepting conformation observed in our crystal structure, the FMN is buried and positioned to accept an electron from redox partner FAD. We propose that the entire FMN domain swings “OUT” from the remainder of the reductase domain to transfer electrons to the active site heme. We are combining scattering data with atomic resolution crystal structures and molecular dynamics simulations to identify snapshots of NOS in solution. We analyze and compare FMN domain movement in different isozymes, making use of overlapping truncated constructs and post-translationally modified proteins. Our SAXS results are consistent with a large swinging movement in the FMN domain that could enable electron transfer from FMN to heme.