A mutation that improves soluble recombinant hemoglobin accumulation in Escherichia coli in heme excess.

High-level expression of soluble recombinant human hemoglobin (rHb) in Escherichia coli was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108-->Lys) in beta-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type beta-globin subunit accumulated. The beta-globin Providence(asp) mutation (Lys-82-->Asp) significantly improved soluble rHb accumulation compared to the wild-type beta-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providenceasp substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-alpha-globin and beta-Lys-82-->Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% +/- 3% of the soluble cell protein in E. coli. The average total accumulation (soluble globin plus insoluble globin) was 56% +/- 7% of the soluble cell protein. Fermentations yielded 6.0 +/- 0. 3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 +/- 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in E. coli depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.

Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli.

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the beta- and di-alpha-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNALeu, similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.

A complete computer monitoring and control system using commercially available, configurable software for laboratory and pilot plant Escherichia coli fermentations.

The advent of inexpensive computers and associated control and data acquisition software makes possible the development of sophisticated, configurable, integrated monitoring and control systems for small-scale laboratory and pilot-scale fermentors at low cost. We describe here the implementation of such a system, the interfacing of off-line instruments to enhance real time data analysis, low level process control and several substrate feeding protocols.

Bacterial nitrite-reducing enzymes.

The enzymic reduction of nitrite takes place in a wide range of bacteria and is found to occur in denitrifying, assimilatory and dissimilatory pathways. In this review we describe the major molecular characteristics of the various enzymes employed in each of these processes.

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.

Studies of the primary oxygen intermediate in the reaction of fully reduced cytochrome oxidase.

The formation and disappearance of a photosensitive species during the reaction of reduced cytochrome c oxidase (putatively a3II.O2), EC 1.9.3.1, has been followed by (a) mixing a3II.CO with O2 in a stopped flow apparatus; (b) initiating the oxygen-oxidase reaction by removing CO with a laser flash; (c) probing the reaction mixture for photosensitivity with a second laser flash. Photosensitivity appears in the reaction mixture after the first laser flash, reaches a maximum after 50-60 microseconds ([O2] greater than 100 microM), and disappears in a further 50-100 microseconds. The kinetics can be represented by the scheme [formula: see text]. In species B, O2 is associated with the protein, possibly CuB, but not with the heme. Species C is the photosensitive a3II.O2 complex, and in D, a3 iron has been oxidized. The formation of species C is responsible for the rapid phase of absorbance change in the oxidase-oxygen reaction. The rate of reaction with oxygen approaches the limit of 35,000 s-1 at high oxygen. Nitric oxide, however, reacts with FeII oxidase with a rate of 1 x 10(8) M-1 s-1, which is accurately maintained up to an observed rate of 10(5) s-1. In flash photolysis experiments, approximately half of the photodissociated nitric oxidase recombines in a biphasic geminate reaction with rates of 1 x 10(8) s-1 and 1 x 10(7) s-1.

Ligand binding in a hierarchy of globin complexes. The hexagonal bilayer hemoglobin of Lumbricus terrestris and its heme-containing subunits.

The kinetics of CO and NO recombination with the giant approximately 3600-kDa hexagonal bilayer hemoglobin of Lumbricus terrestris and its subunits, the approximately 200-kDa dodecamer of globin chains (3 x chains (I + II + III + IV] (see preceding paper (Vinogradov S.N., Sharma, P.K., Qabor, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H., and Gill, S.J. (1991) J. Biol. Chem. 266, 13091-13096], the 50-kDa disulfide-bonded trimer (chains II-IV), the monomer (chain I), and the approximately 30-kDa linker (chains VA, VB, and VI), were measured following photolysis over time scales ranging from picosecond to millisecond. CO recombination at 436 nm subsequent to excitation (9 ns) at 532 nm showed three phases covering a 100-fold range for the Hb, dodecamer, trimer, and linker protein. The proportion of the fast phase was 0.1-0.2 for the trimer, dodecamer, and Hb. The relative rates and amplitudes of the phases were not affected by changes in CO concentration or excitation intensity. The monomer showed a single phase with a rate of 2 x 10(6) M-1 s-1. The second-order reaction with NO showed two rates. The faster rate was 90 x 10(6) M-1 s-1 and accounted for approximately 0.7 of the reaction for all species except the monomer, where it accounted for the full reaction. The slower rate was 15 x 10(6) M-1 s-1 for all species except the monomer.

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant.

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin.

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques.

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)

An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.

The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes.

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.

Ligand binding to a hemoprotein lacking the distal histidine. The myoglobin from aplysia limacina (Val(E7)).

The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme.

Spectroscopic studies of partially reduced forms of Wolinella succinogenes nitrite reductase.

Reductive titrations of the dissimilatory hexa-haem nitrite reductase, Wolinella succinogenes, with methyl viologen semiquinone (MV) and sodium dithionite, have been followed at room temperature by absorption, natural (CD) and magnetic circular dichroism (MCD) spectroscopies and at liquid helium temperature by electron paramagnetic resonance (EPR) and MCD spectroscopies. The nature of the reduced enzyme depends on the reductant employed. At room temperature a single high-spin ferrous haem, observed by MCD after reduction with MV, is absent from dithionite reduced samples. It is suggested that a product of dithionite oxidation becomes bound with high affinity to the reduced state of the enzyme causing the ferrous haem to become low-spin. The site occupied is likely to be the substrate binding haem. The course of the titration with MV at room temperature shows the reduction of high-spin ferric to high-spin ferrous haem. Since the EPR spectrum reveals the presence of an unusual high-low spin ferric haem pair in the oxidised state we propose that the active site of the enzyme is a novel haem pair consisting of one high (5-coordinate) and one low-spin (6 coordinate) haem, magnetically coupled and possibly bridged by a histidinate ligand.

Electron paramagnetic resonance and magnetic circular dichroism studies of a hexa-heme nitrite reductase from Wolinella succinogenes.

The nature of the heme centers in the hexa-heme dissimilatory nitrite reductase from the bacterium Wolinella succinogenes has been investigated with EPR and magnetic circular dichroism spectroscopy. The EPR spectrum of the ferric enzyme is complex showing, in addition to magnetically isolated low-spin ferric hemes with g values of 2.93, 2.3 and 1.48, two sets of signals at g = 10.3, 3.7 and 4.8, 3.21, which we assign to two pairs of exchange coupled hemes. The MCD spectra show that the isolated hemes are bis-histidine coordinated and that there is one high-spin ferric heme. The exchange coupling is lost on treatment with SDS.