Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide.
Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs. (+info)
Subunit dissociation in fish hemoglobins.
The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments. (+info)
Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.
The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride. (+info)
Condensation of carbon in radioactive supernova gas.
Chemistry resulting in the formation of large carbon-bearing molecules and dust in the interior of an expanding supernova was explored, and the equations governing their abundances were solved numerically. Carbon dust condenses from initially gaseous carbon and oxygen atoms because energetic electrons produced by radioactivity in the supernova cause dissociation of the carbon monoxide molecules, which would otherwise form and limit the supply of carbon atoms. The resulting free carbon atoms enable carbon dust to grow faster by carbon association than the rate at which the dust can be destroyed by oxidation. The origin of presolar micrometer-sized carbon solids that are found in meteorites is thereby altered. (+info)
Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10).
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology. (+info)
Heterotropic effectors exert more significant strain on monoligated than on unligated hemoglobin.
The effect of allosteric effectors, such as inositol hexakisphosphate and/or bezafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effectors, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin ( +info)
Evaluation of passive smoking by measuring urinary trans, trans-muconic acid and exhaled carbon monoxide levels.
No method has yet been established to evaluate the exposure to tobacco smoke in passive smoking (PS). We therefore conducted a study on the possibility that the levels of urinary trans, trans-muconic acid (MA) and the exhaled carbon monoxide (CO) could be indices of the passive exposure to tobacco smoke. The moderate correlation was observed between urinary MA levels and the number of consumed cigarettes per day in smokers. The mean urinary MA level of the PS (+) group was significantly higher than that with the PS (-) group. Among the PS (+) group, the mean MA level in the urine obtained in the afternoon was higher than that obtained in the morning. A high correlation was observed between the exhaled CO levels and the number of consumed cigarettes per day in smokers. Like the urinary MA level, the mean exhaled CO level in the PS (+) group, too, gave a significantly higher level than in the PS (-) group. Because the biological half life of MA (7.5 +/- 0.85 h) was longer than that of CO (3.0 +/- 0.36 h), the measurement of urinary MA level is recommended for evaluating the exposure of passive smoking. The measurement of exhaled CO levels is useful only for chain smokers and nonsmokers with PS just before measurement. (+info)
Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands.
We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity. (+info)