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Biomedical subjects

M R Chance

Publications and source records attributed to M R Chance.

At least 37 records · Page 2Linked to original sources
PubMed1997

Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.

Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of .OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 to 100 ms. The sensitivity of DNA and RNA to X-ray mediated .OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-dependent .OH protection. RNA .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from > or = 3.5 to 0.4 min-1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. Synchrotron X-ray footprinting is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.

Animals↗
PubMed1996

Global mapping of structural solutions provided by the extended X-ray absorption fine structure ab initio code FEFF 6.01: structure of the cryogenic photoproduct of the myoglobin-carbon monoxide complex.

X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopic data, including EXAFS, shows that the crystallographic results of Schlichting et al. (1994) are highly relevant to the physiological solution state and must be taken into account in any attempt to understand the incomplete relaxation process of the heme iron for the Mb*CO photoproduct at low temperature.

Animals↗
PubMed1995

Structural and electronic factors that influence oxygen affinities: a spectroscopic comparison of ferrous and cobaltous oxymyoglobin.

Various structural and electronic factors that result in similar rates of oxygen association (kon) and differing rates of oxygen dissociation (koff) for ferrous (FeMb) and cobaltous (CoMb) myoglobin have been investigated. Similar values for kon indicate similar barriers to oxygen binding for CoMb and FeMb. Through optical spectroscopy, we have found that the stable quantum yields of photolysis for CoMbO2 (0.55 +/- 0.05) and FeMbO2 (0.50 +/- 0.05) at 10 K are the same. The X-ray absorption near edge spectra (XANES) of CoMb and FeMb reveal similar metal-heme displacements for the deoxy, oxy, and low temperature photoproduct states of CoMb and FeMb. Thus, similar barriers to ligand binding, indicated by similar kon's and photoproduct yields for CoMb and FeMb, correlate with the metal-heme displacements for the oxy, deoxy, and low temperature photoproduct states of CoMb and FeMb. Lower values of koff for FeMbO2 versus CoMbO2 imply different barriers to oxygen release for the two species. X-ray edge positions of CoMb and FeMb indicate a substantial transfer of electron density from the metal to the ligand upon oxygenation. The distribution of electron density throughout the M-O-O moiety differs for CoMbO2 and FeMbO2. Resonance Raman spectroscopy has demonstrated that the Co-O bond is weaker when compared to Fe-O [Tsubaki, M., & Yu, N. T. (1981) Proc. Natl. Acad. Sci., U.S.A. 78, 3581]. We have used photolyzed/unphotolyzed Fourier Transform Infrared (FTIR) difference spectra of CoMb16O2, CoMb18O2, FeMb16O2, and FeMb18O2 to show that the dioxygen stretching frequency, v(O-O), in CoMbO2 (approximately 1138 cm-1) is higher than FeMbO2 (approximately 1131 cm-1). The dioxygen stretching frequency in CoMbO2 is closer to that of heme protein models lacking a hydrogen bond to the distal histidine, suggesting that formation of the hydrogen bond in FeMbO2 provides a greater effect on the distribution of electron density throughout the Fe-O-O... HN moiety, potentially stabilizing a more ionic Fe-O-O bond. These findings demonstrate important electrostatic differences in the distal environments of CoMbO2 and FeMbO2, resulting in different barriers to oxygen release.

Animals↗
PubMed1995

Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties.

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

Amino Acid Sequence↗
PubMed1995

Structural and electronic factors in heterolytic cleavage: formation of the Co(I) intermediate in the corrinoid/iron-sulfur protein from Clostridium thermoaceticum.

We have completed the first direct structural characterization of an enzyme-bound four-coordinate Co(I) intermediate, in this case for the corrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum. Extended X-ray absorption fine structure and X-ray edge spectroscopy of the active Co(I) state of the C/Fe-SP indicates a four-coordinate (distorted) square-planar structure where the best fit gives average Co-N(equatorial) distances of 1.87 +/- 0.01 A, corresponding to 4.2 +/- 0.3 ligands. The X-ray edge spectrum of Co(I) C/Fe-SP contains a moderate intensity 1s-4p + "shake-down" (SD) transition and no 1s-3d peak (where SD transitions are indicative of square-planar geometries). X-ray edge results for the methyl-Co(III) form, reported earlier [Wirt, M. D., Kumar, M., Ragsdale, S. W., & Chance, M. R. (1993) J. Am. Chem. Soc. 115, 2146-2150], are consistent with a base-off methylcobamide structure. The absence of a ligated 5-methoxybenzimidazole base in the methyl-Co(III) state is important since the base-off form is predicted to predispose the Co-C bond toward heterolytic cleavage to form the four-coordinate Co(I) species concurrent with methyl transfer. Additionally, we have examined first-derivative X-ray edge spectra of Co(I) C/Fe-SP, relative to edge spectra of a cobalt foil, as an indicator of effective nuclear charge on cobalt. The Co(I) C/Fe-SP edge position at 7720.5 +/- 0.3 eV is less than, but very close to, the value seen for the corresponding free Co(I) cobalamin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1994

Kinetic and structural characterization of spinach carbonic anhydrase.

We have carried out kinetics studies of spinach carbonic anhydrase (CA) using stopped-flow spectrophotometry at steady state and 13C-NMR exchange at chemical equilibrium. We found that the rate of CO2<-->HCO3- exchange catalyzed by spinach CA at pH 7.0 to be 3-5 times faster than the maximal kcat for either CO2 hydration or HCO3- dehydration at steady state, suggesting a rate-determining H+ transfer step in the catalytic mechanism. Correspondingly, we measured a pH-independent solvent deuterium isotope effect on kcat of approximately 2.0, and found that the rate of catalysis was significantly decreased at external buffer concentrations below 5 mM. Our results are consistent with a zinc-hydroxide mechanism of action with for spinach CA, similar to that of animal carbonic anhydrases. We have also collected X-ray absorption spectra of spinach CA. Analysis of the extended fine structure (EXAFS) suggests that the coordination sphere of Zn in spinach CA must have one or more sulfur ligands, in contrast to animal CAs which have only nitrogen and oxygen ligands. The models which best fit the data have average Zn-N(O) distances of 1.99-2.06 A, average Zn-S distances of 2.31--2.32 A, and a total coordination number of 4-6. We conclude that animal and spinach CAs are convergently evolved enzymes which are structurally quite different, but functionally equivalent.

Bicarbonates↗
PubMed1994

Sulfur-containing cobalamins: X-ray absorption spectroscopic characterization.

Sulfur-containing cobalamins are thought to have a special role in the intracellular conversion of cyanocobalamin to its coenzyme forms through a Co(I) intermediate. Glutathionylcobalamin is especially interesting as a possible precursor of cobalamin coenzymes [Wagner et al. (1969) Ann. N.Y. Acad. Sci. 112, 580; Pezacka et al. (1990) Biochem. Biophys. Res. Commun. 169, 443]. Recent NMR data [Brown et al. (1993) Biochemistry 32, 8421] strongly support the hypothesis that glutathione coordinates ito the cobalt through the sulfur atom in glutathionylcobalamin. In this study three-sulfur containing cobalamin derivatives (glutathionylcobalamin, sulfitocobalamin, and cysteinylcobalamin) have been characterized by X-ray absorption spectroscopy. We give evidence for the sulfur coordination in these compounds and present the corresponding structural information. The Co-Neq distances are also distances in the sulfur-containing cobalamins are very close to one another (1.90 +/- 0.01 A). The Co-S and Co-Nax distances are also similar (Co-S: 2.28-2.35 A and Co-Nax: 2.13-2.16 A) and in the expected range. The X-ray edge positions for the sulfur derivatives shift to lower energies with respect to cyanocobalamin. This indicates strong electron donation from the sulfur to the cobalt and suggests that the effective charge on the cobalt ion in sulfur cobalamins is largely reduced from +3.

Chemical Phenomena↗
PubMed1993

Human cobalophilin: the structure of bound methylcobalamin and a functional role in protecting methylcobalamin from photolysis.

The interactions of methylcobalamin with cobalophilin from human serum were analyzed using extended X-ray absorption fine structure (EXAFS) spectroscopy, photolysis of the cobalt-carbon bond of methylcobalamin, and a pKa determination of the protonation of the coordinated nitrogen of 5,6-dimethylbenzimidazole (DMB). These results are consistent with the idea that the DMB nitrogen is still coordinated when protein is bound; however, the ability of a methyl radical (generated by photolysis) to escape the geminate cage of the protein is considerably reduced. For methylcobalamin in solution, the DMB nitrogen ligand is at a distance of 2.20 +/- 0.03 A from cobalt [Sagi, I., & Chance, M. R. (1992) J. Am. Chem. Soc. 114, 8061-8066]. This distance to the lower axial ligand does not change when protein binds (2.20 +/- 0.04 A), nor do the optical spectra exhibit any base-off character. The average of the distance from cobalt to the four equatorial nitrogens of the corrin plane is also unchanged. The pKa for the conversion of the "base-on" to the "base-off" form of methylcobalamin, where the above DMB nitrogen becomes protonated and the Co-N axial bond is cleaved, does not deviate from the free cobalamin value of 2.7 when methylcobalamin is bound to cobalophilin. These results indicate that replacement of the DMB ligand with a ligand from the protein is unlikely. Although the background-subtracted EXAFS data sets for free methylcobalamin and for the protein complex are extremely similar, more accurate data with explicit higher shell analysis would be required to entirely rule out ligand replacement. The chemical and electronic nature of the ligand changes little.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Continuous-wave quantum yields of various cobalamins are influenced by competition between geminate recombination and cage escape.

Quantum yields of photolysis of the cobalt-carbon bond for three cobalamin compounds were measured with a continuous-wave laser at 442 nm under both aerobic and anaerobic conditions. Aerobically, the initial homolysis product, Co(II) cobalamin, is trapped by oxygen to form aquocobalamin. Use of an excess of the radical trapping reagent 2,2,6,6-tetramethyl-1-piperidinyloxyl, under anaerobic conditions, scavenges the carbon radical and allows detection of the cobalt(II) photoproduct. Quantum yields measured under anaerobic conditions for 5'-deoxyadenosylcobalamin (phi (Co-C alpha),442 = 0.20 +/- 0.03) and methylcobalamin (phi (Co-C alpha),442 = 0.35 +/- 0.03) are in agreement with the values obtained under aerobic conditions (phi (Co-C alpha),442 = 0.19 +/- 0.04 and phi (Co-C alpha),442 = 0.36 +/- 0.04, respectively). Additionally, the quantum yield values for 5'-deoxyadenosylcobalamin and its base-off derivative (phi (Co-C alpha),442 = 0.045 +/- 0.015) match those obtained on a nanosecond time scale [Chen, E., & Chance, M. R. (1990) J. Biol. Chem. 256, 12987-12994]. A comparison of quantum yields obtained anaerobically for 5'-deoxyadenosylcobalamin and methylcobalamin in H2O versus ethylene glycol shows a 4-fold decrease for the former cobalamin and no change for the latter. These quantum yields are evaluated in terms of time-independent radical separation distances.

Aerobiosis↗
PubMed1993

Temperature dependent coordination effects in base-off adenosyl and methylcobalamin by X-ray edge spectroscopy.

Examination of the role of base-off cobalamin species (where the 5,6-dimethylbenzimidazole ligand coordinated to cobalt is detached by protonation of the imidazole nitrogen) in differentiation between homolytic and heterolytic cobalt-carbon bond cleavage mechanisms is a primary step in better understanding B12-dependent enzyme catalysis. X-ray absorption edge spectroscopy provides the first direct structural evidence of five-coordination in base-off adenosyl- and base-off methylcobalamin complexes at room temperature. Integration of 1s-3d pre-edge transitions of the base-off species reveals the dependence of coordination number on temperature. Gradual increases in 1s-3d transition intensities, as the temperature is increased from 180 K to 298 K, reflect a change in the coordination number from six (where a water molecule is presumed to occupy the coordination site vacated by the 5,6-dimethylbenzimidazole ligand) to primarily five-coordinate. Base-off configurations that strengthen the Co-C bond may be both decreasing the tendency for homolytic cleavage while increasing the tendency for hetrolytic Co(I) B12 formation.

Cobamides↗
PubMed1992

Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc.

Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.

Absorptiometry, Photon↗
PubMed1992

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

Amino Acid Sequence↗
PubMed1992

Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis.

X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.

Cobamides↗
PubMed1991

Identification of structural markers for vitamin B12 and other corrinoid derivatives in solution using FTIR spectroscopy.

The identification of structural markers for B12/protein interactions is crucial to a complete understanding of vitamin B12 transport and metabolic reaction mechanisms of B12 coenzymes. Fourier transform infrared spectroscopy can provide direct measurements of changes in the side chains and corrin ring resulting from B12/protein interactions. Using FTIR spectroscopy in various solvent systems, we have identified structural markers for corrinoids in the physiological state. We assign the major band (denoted B), which occurs at ca. 1630 cm-1 in D2O and ca. 1675 cm-1 in ethanol, to the amide I C=O stretching mode of the propionamide side chains of the corrin ring. The lower frequency of band B in D2O versus ethanol is due to the greater hydrogen-bonding properties of D2O that stabilize the charged amide resonance form. Since the propionamides are known to be important in protein binding, band B is a suitable marker for monitoring the interaction of these side chains with proteins. We assign bands at ca. 1575 and 1545 cm-1 (denoted C and D) as breathing modes of the corrin ring on the basis of the bands' solvent independence and their sensitivity to changes in axial ligation. As the sigma-donating strength of the axial ligands increases, the frequencies of bands C and D decrease, possibly indicating a lengthening of the corrin conjugated system. Band A, the known cyanide stretching frequency at ca. 2130 cm-1, probes the cobalt-carbon distance in cyanocorrinoids. As the frequency of band A increases, the cobalt-carbon bond strength should decrease.

Amides↗
PubMed1990

Nanosecond transient absorption spectroscopy of coenzyme B12. Quantum yields and spectral dynamics.

Photolysis of adenosylcobalamin leads to homolytic cleavage, similar to many of the B12-dependent enzyme reactions. Therefore, we have used photolysis to study the structure and lability of the cobalt-carbon bond. The nanosecond quantum yield for adenosylcobalamin is 0.23 +/- 0.04, higher than reported previously. The acidified form of adenosylcobalamin, so called "base-off" B12, has a much lower quantum yield at 0.045 +/- 0.015, demonstrating an inverse correlation between cobalt-carbon bond strength and quantum yield. Investigation of the wavelength dependence of the quantum yield shows that there is a highly efficient transmission of energy from the corrin ring to the cobalt-carbon bond. A comparison of nanosecond transient and static spectra showed small spectral differences. Therefore, any spectral relaxation of a sterically distorted corrin ring may be detectable only at sub-nanosecond timescales. Spectral analysis also provides data on the kinetics of recombination. In the absence of enzyme, geminate rebinding must be substantial, since the rate of Co(II) and deoxyadenosyl radical recombination is near the diffusion controlled limit. Therefore, it is likely that the enzyme functions to pull the geminate partners apart, perhaps as suggested previously, through a conformational change. The importance of geminate recombination in the mechanism of homolytic cleavage is further supported by a comparison of our results with picosecond transient absorption studies.

Cobamides↗
PubMed1990

O2 and CO reactions with heme proteins: quantum yields and geminate recombination on picosecond time scales.

Picosecond time-resolved absorption spectroscopy and low-temperature studies have been undertaken in order to understand the nature of the intrinsic quantum yields and geminate recombination of carbon monoxide and oxygen to hemoglobin and myoglobin. We find that the photoproduct yields at 40 ps and long times (minutes) after photolysis at 8 K are similar; however, the yield of oxygen photoproducts is 0.4 +/- 0.1 while the yield of carbon monoxide photoproducts is 1.0 +/- 0.1 for both myoglobin and hemoglobin. Measurements in the Soret, near-infrared, and far-IR are used to quantitate the photoproduct yields. These results call into question previous cryogenic kinetic studies of O2 recombination. Significant subnanosecond geminate recombination is observed in oxyhemoglobin down to 150 K, while below 100 K this geminate recombination disappears. The lower photoproduct yields for oxyheme protein complexes can be attributed to both subnanosecond and subpicosecond recombination events which are ligand and protein dynamics dependent.

Animals↗