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1.  CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier 
The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation. The ligation states of the heme under different solvent conditions are also investigated using both equilibrium and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylammonium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes. In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with rapid (10–100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, Hp, to be 11 ± 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy barriers: ΔGD ≈ 12 ± 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes in the distal pocket and the xenon-4 cavity of myoglobin (~3 kJ/mol), then lead to a distal pocket enthalpic barrier of HD ≈ 9 ± 2 kJ/mol. These results agree well with the predictions of a simple model and with previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the enthalpic MbCO rebinding barrier (18 ± 2 kJ/mol).
doi:10.1021/ja042365f
PMCID: PMC2768272  PMID: 15839683
2.  Responses of Mycobacterium tuberculosis Hemoglobin Promoters to In Vitro and In Vivo Growth Conditions▿  
Applied and Environmental Microbiology  2008;74(11):3512-3522.
The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H2O2, but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ∼2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased ∼2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host.
doi:10.1128/AEM.02663-07
PMCID: PMC2423021  PMID: 18390674
3.  Vibrational Coherence Spectroscopy of the Heme Domain in the CO-Sensing Transcriptional Activator CooA 
Journal of the American Chemical Society  2011;133(46):18816-18827.
Femtosecond vibrational coherence spectroscopy was used to investigate the low frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (kBT = 200 cm-1) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm-1 in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm-1 in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm-1 that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm-1). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm-1 in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm-1 disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated and, although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.
doi:10.1021/ja206152m
PMCID: PMC3218251  PMID: 21961804
Ch-CooA; DNA-Protein interaction; low frequency modes; heme distortion; Fe-His stretching vibration
4.  Modulating Distal Cavities in the α and β Subunits of Human HbA Reveals the Primary Ligand Migration Pathway† 
Biochemistry  2011;50(34):7361-7374.
The free volume in the active site of human HbA plays a crucial role in governing the bimolecular rates of O2, CO, and NO binding, the fraction of geminate ligand recombination, and the rate of NO dioxygenation by the oxygenated complex. We have decreased the size of the distal pocket by mutating Leu(B10), Val(E11) and Leu(G8) to Phe and Trp and of other more internal cavities by filling them with Xe at high gas pressures. Increasing the size of the B10 side chain reduces bimolecular rates of ligand binding nearly 5,000-fold and inhibits CO geminate recombination due to both reduction of the capture volume in the distal pocket and direct steric hindrance of Fe-ligand bond formation. Phe and Trp(E11) mutations also cause a decrease in distal pocket volume but, at the same time, increase access to the Fe atom due to the loss of the γ2 CH3 group of the native Val(E11) side chain. The net result of these E11 substitutions is a dramatic increase in geminate recombination because dissociated CO is sequestered close to the Fe atom and can rapidly rebind without steric resistance. However, the bimolecular rate constants for ligand binding to the Phe and Trp(E11) mutants are decreased 5–30-fold, due to a smaller capture volume. Geminate and bimolecular kinetic parameters for Phe and Trp(G8) mutants are similar to those for the native HbA subunits because the aromatic rings at this position cause little change in distal pocket volume and because ligands do not move past this position into the globin interior of wild-type HbA subunits. The latter conclusion is verified by the observation that Xe binding to the α and β Hb subunits has little effect on either geminate or bimolecular ligand rebinding. All of these experimental results argue strongly against alternative ligand migration pathways that involve movements through the protein interior in HbA. Instead, ligands appear to enter through the His(E7) gate and are captured directly in the distal cavity.
doi:10.1021/bi200923k
PMCID: PMC3160505  PMID: 21793487
hemoglobin; myoglobin; xenon binding sites; geminate recombination; flash photolysis; ligand migration pathways; non-covalent binding site
5.  Role of Copper Ion in Regulating Ligand Binding in a Myoglobin-Based Cytochrome c Oxidase Model 
Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a3) and a copper atom (CuB) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named CuBMb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in CuBMb was investigated. Resonance Raman studies show that the presence of copper in CO-bound CuBMb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu+; it subsequently transfers from Cu+ to Fe2+ in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu+ and the slow intramolecular CO transfer rate between Cu+ and Fe2+ in the CuBMb/Cu+ complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound CuBMb/Cu+ complex, the photolyzed ligand transiently binds to Cu+ and subsequently rebinds to Fe2+, accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the CuBMb/Cu+ complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.
doi:10.1021/ja907777f
PMCID: PMC3761084  PMID: 20070118
6.  Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering 
Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I1, I2, and I3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I1 intermediate is generated within 100 ps and transforms to the R-like I2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I3 intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.
doi:10.1021/ja210856v
PMCID: PMC3337689  PMID: 22494177
7.  Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy 
Biochimica et biophysica acta  2010;1797(9):1657-1664.
Summary
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully-reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully-reduced enzyme. The difference demonstrates that in the fully-reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully-reduced enzyme monoexponentially with τ ~12 µs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ~14 ns, 14 µs, and 280 µs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully-reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14 µs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280 µs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.
doi:10.1016/j.bbabio.2010.05.010
PMCID: PMC3990236  PMID: 20529691
respiration; chlorin; cytochrome; ligand binding; gas molecule; photobiology
8.  Temperature dependent studies of NO recombination to heme and heme proteins 
Journal of the American Chemical Society  2005;127(48):16921-16934.
The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200K, the transition state enthalpy barrier associated with the fastest (~10ps) recombination phase is found to be zero, while a slower geminate phase (~200ps) reveals a small enthalpic barrier (~ 3 ± 1 kJ/mol). Both of the kinetic rates slow down slightly in the myoglobin (Mb) samples above 200K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (~200ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) NO rebinding process observed below 200K is independent of the presence or absence of the proximal histidine ligand. In contrast, the slowing of the kinetic rates above 200K in MbNO disappears in the absence of the protein. Generally, the data indicate that, in contrast to CO, the NO ligand binds to the heme iron through a “harpoon” mechanism where the heme iron out-of-plane conformation presents a negligible enthalpic barrier to NO rebinding. These observations strongly support a previous analysis (J. Am. Chem. Soc. 1988, 110, 6656) that primarily attributes the low temperature stretched exponential rebinding of MbCO to a quenched distribution of heme geometries. A simple model is presented for MbNO rebinding that explains a variety of experiments, including the dependence of the kinetic amplitudes on the pump photon energy.
doi:10.1021/ja054249y
PMCID: PMC2553725  PMID: 16316238
9.  Evolution, Three-Dimensional Model and Localization of Truncated Hemoglobin PttTrHb of Hybrid Aspen 
PLoS ONE  2014;9(2):e88573.
Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.
doi:10.1371/journal.pone.0088573
PMCID: PMC3919811  PMID: 24520401
10.  Photolysis of Adenosylcobalamin and Radical Pair Recombination in Ethanolamine Ammonia-Lyase Probed on the Micro- to Millisecond Time Scale by using Time-Resolved Optical Absorption Spectroscopy† 
Biochemistry  2009;48(1):140-147.
The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl) in coenzyme B12 (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10-7 - 10-1 s time scale at 295 K by using transient ultraviolet-visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin-5′-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt-carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10-7 s for cob(II)alamin of 0.08 ±0.01, which is 3-fold less than for AdoCbl in aqueous solution (0.23 ±0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, Pf, Ps, and Pc, are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: Pf (2.2×103 s-1; 0.02), Ps (4.2×102 s-1; 0.01), and Pc (constant amplitude, no recombination; 0.05). Binding of the substrate analog, (S)-1-amino-2-propanol, to EAL eliminates the Pf state, and lowers the quantum yield of Pc (0.03) relative to Ps (0.01), but does not significantly change the quantum yield or decay rate constant of Ps, relative to holo-EAL. The substrate analog thus influences the quantum yield at 10-7 s by changing the cage escape rate from the geminate cob(II)alamin-5′-deoxyadenosyl radical pair state. However, the predicted substrate analog binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analog does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.
doi:10.1021/bi801659e
PMCID: PMC2642536  PMID: 19072291
11.  Cloning and Heterologous Expression of a β-d-Mannosidase (EC 3.2.1.25)-Encoding Gene from Thermobifida fusca TM51 
Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, β-xylosidases, endomannanases, and β-mannosidases, when grown on cellulose or hemicellulose as carbon sources. β-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for β-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a β-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative β-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only β-d-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl β-d-mannopyranoside (pNP-βM) substrate were as follows: Km = 180 μM and Vmax = 5.96 μmol min−1 mg−1; the inhibition constant for mannose was Ki = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-αM and pNP-βM; under these conditions mannosyl groups were transferred by the enzyme from pNP-βM to pNP-αM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.
doi:10.1128/AEM.69.4.1944-1952.2003
PMCID: PMC154781  PMID: 12676668
12.  Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3) 
Background
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.
Results
T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase.
Conclusions
In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.
doi:10.1186/1475-2859-11-8
PMCID: PMC3286373  PMID: 22239833
alpha-hemolysin secretion pathway; cutinase; protein secretion; extracellular production
13.  Investigations of Ferric Heme Cyanide Photodissociation in Myoglobin and Horseradish Peroxidase 
The journal of physical chemistry. B  2013;117(15):4042-4049.
The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has been definitively observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881–1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252–2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75% and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate kBA ≈ (3.6 ps)−1 is the dominant process, some CN− exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN− association rate, we find that the CN− escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ≈ 1–2 × 107 s−1. This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1×107 s−1) under the same conditions. The analysis leads to an escape probability kout/(kout+kBA) ~ 10−4, which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN− bimolecular association rate (170 M−1s−1), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN− bimolecular association rate is larger by ~103 making the CN− photolysis more difficult to observe.
doi:10.1021/jp401224f
PMCID: PMC3631277  PMID: 23472676
Myoglobin CN; heme cyanide; photolysis; resonance Raman
14.  Linking Conformation Change to Hemoglobin Activation Via Chain-Selective Time-resolved Resonance Raman Spectroscopy on Protoheme/Mesoheme Hybrids 
Time-resolved Resonance Raman spectra are reported for Hb tetramers, in which the αand β chains are selectively substituted with mesoheme. The Soret absorbtion band shift in meso- relative to proto-heme permits chain-selective excitation of heme RR spectra. The evolution of these spectra following HbCO photolysis show that geminate recombination rates and yields are the same for the two chains, consistent with recent results on 15N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxy-heme ν4 and νFe-His) RR bands, which are anti-correlated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UVRR spectra. Both chains show Fe-His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, Rdeoxy (0.07 μs), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe-His bond weakens, more so for the α than the β chains. The weakening is gradual for the β chains, but abrupt for the α chains, coinciding with completion of the R-T quaternary transition, at 20μs. Since the transition from fast- to slow-rebinding Hb also occurs at 20μs, the drop in the α chain νFe-His supports the localization of ligation restraint to tension in the Fe-His bond, at least in the α-chains. The mechanism is more complex in the β chains.
doi:10.1007/s00775-009-0487-7
PMCID: PMC2880192  PMID: 19288145
Protoheme/mesoheme; hybrid hemoglobin; resonance Raman; geminate recombination; allostery
15.  Coexpression of Human α- and Circularly Permuted β-Globins Yields a Hemoglobin with Normal R State but Modified T State Properties† 
Biochemistry  2009;48(23):5456-5465.
For the first time, a circularly permuted human β-globin (cpβ) has been coexpressed with human α-globin in bacterial cells and shown to associate to form α-cpβ hemoglobin in solution. Flash photolysis studies of α-cpβ show markedly biphasic CO and O2 kinetics with the amplitudes for the fast association phases being dominant due the presence of large amounts of high-affinity liganded hemoglobin dimers. Extensive dimerization of liganded but not deoxygenated α-cpβ was observed by gel chromatography. The rate constants for O2 and CO binding to the R state forms of α-cpβ are almost identical to those of native HbA (k′R(CO) ≈ 5.0 μM−1 s−1; k′R(O2) ≈ 50 μM−1 s−1), and the rate of O2 dissociation from fully oxygenated α-cpβ is also very similar to that observed for HbA (kR(O2) ≈ 21–28 s−1). When the equilibrium deoxyHb form of α-cpβ is reacted with CO in rapid mixing experiments, the observed time courses are monophasic and the observed bimolecular association rate constant is ∼1.0 μM−1 s−1, which is intermediate between the R state rate measured in partial photolysis experiments (∼5 μM−1 s−1) and that observed for T state deoxyHbA (k′T(CO) ≈ 0.1 to 0.2 μM−1 s−1). Thus the deoxygenated permutated β subunits generate an intermediate, higher affinity, deoxyHb quaternary state. This conclusion is supported by equilibrium oxygen binding measurements in which α-cpβ exhibits a P50 of ∼1.5 mmHg and a low n-value (∼1.3) at pH 7, 20 °C, compared to 8.5 mmHg and n ≈ 2.8 for native HbA under identical, dilute conditions.
doi:10.1021/bi900216p
PMCID: PMC2725443  PMID: 19397368
16.  Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode▿  
Applied and Environmental Microbiology  2009;75(23):7335-7342.
We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
doi:10.1128/AEM.01241-09
PMCID: PMC2786427  PMID: 19820154
17.  Modulation of the Heme Electronic Structure and Cystathionine β-synthase Activity by Second Coordination Sphere Ligands: The Role of Heme Ligand Switching in Redox Regulation† 
Journal of inorganic biochemistry  2009;103(5):689-697.
In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424 nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449 nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is −350 ± 4 mV and is substantially lower than the value of −287 ± 2 mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.
doi:10.1016/j.jinorgbio.2009.01.009
PMCID: PMC2772092  PMID: 19232736
18.  Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms 
Biochimica et biophysica acta  2010;1814(3):405-408.
The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H4B) cofactor and L-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of L-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.
doi:10.1016/j.bbapap.2010.11.007
PMCID: PMC3039037  PMID: 21146639
Nitric oxide synthase; Kinetics; Mutation; Mechanism; Ligand binding
19.  A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily 
DyP-type peroxidases comprise a novel superfamily of heme-containing peroxidases which is unrelated to the superfamilies of known peroxidases and of which only a few members have been characterized in some detail. Here, we report the identification and characterization of a DyP-type peroxidase (TfuDyP) from the thermophilic actinomycete Thermobifida fusca. Biochemical characterization of the recombinant enzyme showed that it is a monomeric, heme-containing, thermostable, and Tat-dependently exported peroxidase. TfuDyP is not only active as dye-decolorizing peroxidase as it also accepts phenolic compounds and aromatic sulfides. In fact, it is able to catalyze enantioselective sulfoxidations, a type of reaction that has not been reported before for DyP-type peroxidases. Site-directed mutagenesis was used to determine the role of two conserved residues. D242 is crucial for catalysis while H338 represents the proximal heme ligand and is essential for heme incorporation. A genome database analysis revealed that DyP-type peroxidases are frequently found in bacterial genomes while they are extremely rare in other organisms. Most of the bacterial homologs are potential cytosolic enzymes, suggesting metabolic roles different from dye degradation. In conclusion, the detailed biochemical characterization reported here contributes significantly to our understanding of these enzymes and further emphasizes their biotechnological potential.
doi:10.1007/s00253-009-2369-x
PMCID: PMC2854361  PMID: 19967355
Peroxidase; Heme; Sulfoxidation; Enantioselective; Dye decolorizing
20.  Quantitative Modeling Assesses the Contribution of Bond Strengthening, Rebinding and Force Sharing to the Avidity of Biomolecule Interactions 
PLoS ONE  2012;7(9):e44070.
Cell adhesion is mediated by numerous membrane receptors. It is desirable to derive the outcome of a cell-surface encounter from the molecular properties of interacting receptors and ligands. However, conventional parameters such as affinity or kinetic constants are often insufficient to account for receptor efficiency. Avidity is a qualitative concept frequently used to describe biomolecule interactions: this includes incompletely defined properties such as the capacity to form multivalent attachments. The aim of this study is to produce a working description of monovalent attachments formed by a model system, then to measure and interpret the behavior of divalent attachments under force. We investigated attachments between antibody-coated microspheres and surfaces coated with sparse monomeric or dimeric ligands. When bonds were subjected to a pulling force, they exhibited both a force-dependent dissociation consistent with Bell’s empirical formula and a force- and time-dependent strengthening well described by a single parameter. Divalent attachments were stronger and less dependent on forces than monovalent ones. The proportion of divalent attachments resisting a force of 30 piconewtons for at least 5 s was 3.7 fold higher than that of monovalent attachments. Quantitative modeling showed that this required rebinding, i.e. additional bond formation between surfaces linked by divalent receptors forming only one bond. Further, experimental data were compatible with but did not require stress sharing between bonds within divalent attachments. Thus many ligand-receptor interactions do not behave as single-step reactions in the millisecond to second timescale. Rather, they exhibit progressive stabilization. This explains the high efficiency of multimerized or clustered receptors even when bonds are only subjected to moderate forces. Our approach provides a quantitative way of relating binding avidity to measurable parameters including bond maturation, rebinding and force sharing, provided these parameters have been determined. Also, this provides a quantitative description of the phenomenon of bond strengthening.
doi:10.1371/journal.pone.0044070
PMCID: PMC3443103  PMID: 23024747
21.  Comparative Analysis of Mycobacterial Truncated Hemoglobin Promoters and the groEL2 Promoter in Free-Living and Intracellular Mycobacteria 
Applied and Environmental Microbiology  2012;78(18):6499-6506.
The success of Mycobacterium tuberculosis depends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO2 levels. Therefore, an effective detoxification system is required for the pathogen to persist in vivo. The genome of M. tuberculosis contains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by the glbO and glbN genes, respectively, important in the survival of M. tuberculosis in macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. Whereas glbN showed an early response to the oxidative and nitrosative stresses tested, glbO gave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested, groEL2 showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb and groEL2 mRNAs showed an initial upregulation at 24 h postinfection. The presence of the glbO protein imparted an increased survival to M. smegmatis in THP-1 differentiated macrophages compared to that imparted by the glbN and hsp65 proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival of M. tuberculosis in the hostile environment of the host.
doi:10.1128/AEM.01984-12
PMCID: PMC3426685  PMID: 22773641
22.  Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle 
The Journal of General Physiology  1991;98(4):657-679.
Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross- bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179- 192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.
PMCID: PMC2229081  PMID: 1960528
23.  Hyper secretion of Thermobifida fusca β-glucosidase via a Tat-dependent signal peptide using Streptomyces lividans 
Background
Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans.
Results
Three candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide.
Conclusion
In the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.
doi:10.1186/1475-2859-12-88
PMCID: PMC3850917  PMID: 24083334
Streptomyces; Protein secretion; Signal peptide sequence; Tat pathway
24.  Dynamics of Carbon Monoxide Binding to Cystathionine β-Synthase 
The Journal of biological chemistry  2006;281(19):13433-13438.
Cystathionine β-synthase (CBS), condenses homocysteine, a toxic metabolite, with serine, in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor, to which CO or NO can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding, with successive association constants of 0.24 and 0.02 μM−1. Stopped-flow measurements reveal slow CO association (0.0166 s−1) limited by dissociation of the endogenous ligand, Cys52. Rebinding of CO and of Cys52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys52 rebinding rate, 4000 s−1, is essentially unchanged between pH 7.6 and 10.5, indicating that the pKa of Cys52 is shifted below pH 7.6. This effect is attributed to the nearby Arg266 residue, which is proposed to form a salt-bridge with the dissociated Cys52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt-bridge suggests a pathway for enzyme inactivation upon CO binding, since Arg266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.
doi:10.1074/jbc.M600246200
PMCID: PMC2745537  PMID: 16505479
25.  Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains 
Applied and Environmental Microbiology  2013;79(17):5159-5166.
The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
doi:10.1128/AEM.01214-13
PMCID: PMC3753941  PMID: 23793636

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