Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin.

We report the utility of native-state mass spectrometry to detect zinc ion binding to the engineered hemoglobin rHb52. Various preparations of this recombinant hemoglobin had significantly different oxygen affinities. Detailed characterization of denatured globins did not show any difference between analyzed hemoglobin molecules. However, when solutions of intact hemoglobin pseudotetramers were analyzed by native-state electrospray mass spectrometry, a significant shift in the mass spectrum was observed, indicating labile modification of hemoglobin. Using collision-induced dissociation (CID), we found a mass gain of 63 Da located on the beta-globin. EDTA treatment of modified hemoglobin prior to the infusion removed the modification and restored the predicted oxygen affinity. Ion-trap fragmentation of the +8 charged ion of modified beta-globin showed a neutral loss of 96+/-1 Da, consistent with neutral loss of zinc sulfide. These findings indicated zinc binding to the beta-globin through a cysteine residue. Involvement of Cys93 was confirmed by kinetics of cysteine residue reactivity with dithiodipyridine and peptide mapping. Presence of zinc was confirmed by ICP-MS metal analysis.

Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin.

Administration of extracellular hemoglobin-based oxygen carriers often induces mild increases in blood pressure. In order to test whether nitric oxide (NO) scavenging is responsible for the hypertensive effect, we constructed and tested a set of recombinant hemoglobins that vary in rates of reaction with NO. The results suggest that the rapid reactions of oxy- and deoxyhemoglobin with nitric oxide are the fundamental cause of the hypertension. The magnitude of the blood-pressure effect correlates directly with the in vitro rate of NO oxidation. Hemoglobins with decreased NO-scavenging activity may be more suitable for certain therapeutic applications than those that cause depletion of nitric oxide.

Mechanism of NO-induced oxidation of myoglobin and hemoglobin.

Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

The gateway to the active site of heme-copper oxidases.

The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases. The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels. CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway. FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex. Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO. Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme. These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site. Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site. We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes.

Photodissociation and recombination of carbonmonoxy cytochrome oxidase: dynamics from picoseconds to kiloseconds.

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)

Interdomain salt bridges modulate ligand-induced domain motion of the sulfate receptor protein for active transport.

The refined crystal structure of the liganded form of the Salmonella typhimurium sulfate-binding protein, a periplasmic receptor of active transport, is made up of two globular domains bisected by a deep cleft wherein the dehydrated sulfate is completely engulfed and bound by hydrogen bonds and van der Waals' forces. Two salt bridges (between Glu15 and Arg174 and between Asp68 and Arg134) span the cleft opening. To elucidate the role of the inter-domain salt bridges in the ligand-induced domain motion, the acidic residues were changed (singly and together) to their corresponding amide side-chains by site-directed mutagenesis of the recombinant Escherichia coli sulfate-binding protein. Rapid kinetics and equilibrium measurements of sulfate binding to the purified mutant proteins demonstrate that these salt bridges stabilize the closed liganded form of the receptor and modulate the rate of cleft opening. Our results have new implications in understanding the dynamics of many other multidomain proteins that undergo similar large-scale domain motions.

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.

Using recombinant DNA techniques, an Escherichia coli periplasmic sulfate receptor or sulfate-binding protein involved in active transport has been overexpressed and characterized. This protein is essentially identical in size, sequence, antigenicity, and ligand affinity and specificity to the sulfate receptor from Salmonella typhimurium whose crystal structure has been refined at 2 A resolution. The dehydrated sulfate is bound in the deep cleft between the two lobes of the bilobate protein. Using the structure of the S. typhimurium as a guide, three site-directed mutants (Ser129Cys, Gly46Cys, and Ser129Cys/Gly46Cys) have been made. In the Cys129/Cys46 mutant the disulfide has been successfully introduced across the opening of the ligand-binding site cleft of the E. coli sulfate-binding protein. The dissociation of sulfate from the double mutant protein is very slow under oxidizing conditions and increases more than 200-fold when reducing agent is added. This effect is attributed to a loss of interdomain structural flexibility in the presence of the disulfide, and underscores the importance of protein conformational change in binding protein function.

A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies.

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.

An in vitro capillary system for studies on microcirculatory O2 transport.

An in vitro artificial capillary system has been developed for use in examining the O2 transport properties of free hemoglobin and erythrocytes. The artificial capillary was constructed by casting a thin film of transparent silicone rubber around a strand of tungsten wire that was 24 micron in diameter. After the rubber had polymerized, the wire was removed. Typical dimensions of the silicone rubber film were 170 micron thick, 1 cm wide, 5 mm long in the direction of flow, and a 27-micron lumen diameter. The artificial capillary bed was mounted on a microscope and perfused by either hemoglobin solutions or cell suspensions. Fractional saturation was measured as a function of axial position by a dual-wave-length microspectrophotometer, and the flow rate was regulated precisely by a syringe pump. O2 release experiments were carried out by suffusing the gas space surrounding the artificial capillary film with 100% N2 and perfusing with an oxygenated sample. O2 uptake experiments were carried out by suffusing the gas space with O2-N2 mixtures and perfusing with deoxygenated samples. The axial velocities were varied from 3 to 15 mm/s. The residence time (the time a particular red cell or hemoglobin molecule has spent in the capillary) for 50% oxygenation of a 4 mM (heme) deoxyhemoglobin solution was approximately 0.05 s at 37 degrees C when the gas space surrounding the capillary contained air. The corresponding time for 50% oxygenation of an equivalent red cell suspension was approximately 0.25 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological factors affecting O2 transport by hemoglobin in an in vitro capillary system.

O2 transport was examined by measuring the fractional saturation of concentrated hemoglobin solutions flowing through an artificial capillary that was approximately 27 micron in diameter and embedded in a silicone rubber film approximately 170 micron thick. The effects of pH, hemoglobin concentration, O2 tension, temperature, and organic phosphate were measured and analyzed quantitatively by a rigorous mathematical model that included the geometry of the capillary in the silicone film, parabolic flow velocity distributions inside the lumen, and cooperative O2 binding by hemoglobin. The rates of both oxygenation and deoxygenation were limited by diffusion and governed by the magnitude of the O2 gradient between the intracapillary fluid phase and the external gas space. In uptake experiments, O2 flux is determined primarily by the external O2 tension (16-160 mmHg in our experiments) because the internal O2 pressure is kept small due to chemical combination with hemoglobin. In release experiments, the external O2 tension is maintained at zero, and the transport rate is determined by the intracapillary partial pressure of O2 that is proportional to the O2 half-saturation pressure of hemoglobin value of the hemoglobin sample. As a result, factors that change the affinity of hemoglobin for O2, such as pH, temperature, and organic phosphate concentration, influence strongly the rate of O2 release but have little effect on the rate of O2 uptake. These properties are physiologically advantageous, since a decrease in pH or an increase in temperature during exercise increases both the rate and extent of deoxygenation while not altering the kinetics of oxygenation.