Biomedical subjects

D Beece

Publications and source records attributed to D Beece.

5 recordsLinked to original sources

PubMed1984

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. II. Quantitation and preliminary modeling of the M----bR reactions.

The cell membrane of Halobacterium halobium (H. halobium) contains the proton-pump bacteriorhodopsin, which generates a light-driven transmembrane protonmotive force. The interaction of the bacteriorhodopsin photocycle with the electric potential component of the protonmotive force has been investigated. H. halobium cell envelope vesicles have been prepared by sonication and further purified by ultracentrifugation on Ficoll/NaCl/CsCl density gradients. Under continuous illumination (550 +/- 50 nm) varied from 0 to 40 mW cm-2, the vesicles maintain a membrane potential of 0 to -100 mV. The membrane potential was measured by flow dialysis of 3H-TPMP+ uptake and could be abolished by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Time-resolved absorption spectroscopy was used to measure the decay kinetics of the M photocycle intermediate, which was initiated by a weak laser flash (588 nm), while the vesicles were continuously illuminated as above. The M decay kinetics were fitted with two exponential decays by a computer deconvolution program. The faster decaying form decreases in amplitude (70 to 10% of the total) and the slower decaying form increases in amplitude and lifetime (23 to 42 ms) as the background light intensity increases. Although any correlation between the membrane potential and the bacteriorhodopsin photocycle M-forms is complex, the present data will allow specific tests of the physical mechanism for this interaction to be designed and conducted.

Bacteriorhodopsins↗

PubMed1982

Control and pH dependence of ligand binding to heme proteins.

The recombination after flash photolysis of dioxygen and carbon monoxide with sperm whale myoglobin (Mb), and separated beta chains of human hemoglobin (beta A) and hemoglobin Zürich (beta ZH), has been studied as a function of pH and temperature from 300 to 60 K. At physiological temperatures, a preequilibrium is established between the ligand molecules in the solvent and in the heme pocket. The ligand in the pocket binds to the heme iron by overcoming a barrier at the heme. The association rate is controlled by this final binding step. The association rate of CO to Mb and beta A is modulated by a single titratable group with a pK at 300 K of 5.7. The binding of CO to beta ZH, in which the distal histidine is replaced by arginine, does not depend on pH. Oxygen recombination is independent of pH in all three proteins. Comparison of the binding of CO at 300 K and at low temperatures shows that pH does not affect the preequilibrium but changes the barrier height at the heme. The pH dependence and the difference between O2 and CO binding can be explained by a charge-dipole interaction between the distal histidine and CO.

Adult↗

PubMed1982

Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures.

We have studied the infrared spectra of the bound and photodissociated states of Mb-12CO and Mb-13CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-12CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-1. The low-temperature photodissociated state (Mb) shows three lines (B0, B1, B2) at 2144, 2131, and 2119 cm-1 for 12CO. The absorbances of the three lines depend on temperature. B0 is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb have near-infrared, EPR, and Mössbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.

Animals↗

PubMed1980

Solvent viscosity and protein dynamics.

Proteins are dynamic systems. Recent evidence demonstrates that they exist in a large number of conformational substates and can continuously move from one substate to another; motion of a small ligand inside a protein may be possible only through these conformational fluctuations. To test this idea, we study with flash photolysis the binding of CO to protoheme and O2 and CO to myoglobin in many different solvents. The standard evaluation of such experiments yields information only about the protein-solvent system. A novel approach is presented which permits conclusions concerning the protein: Data from all solvents are considered together, and the rates for transitions of the ligand over various barriers are studied as a function of temperature for fixed solvent viscosities. Results show that over a wide range in viscosity the transition rates in heme-CO are inversely proportional to the solvent viscosity and can consequently be described by the Kramers equation. The rates of O2 and CO in myoglobin also depend on the solvent viscosity and are most sensitive to the solvent at the lowest viscosity. Viscosity influences protein reactions even in aqueous solutions. The data dan be interpreted by a dynamic model in which transitions into and inside myoglobin are governed by fluctuations between conformational substates corresponding to closed and open pathways. Ligand motion thus is mainly controlled by gates and not by static potential barriers. Some characteristic parameters for the substates are determined, and they agree approximately with similar parameters found in Mössbauer experiments. As expected, the barrier parameters evaluated in the novel approach deviate markedly from the ones obtained by the conventional procedure. Comparison with model calculations or basic theories will be meaningful only with the new evaluation, and the method may be essential for many or possibly all biochemical reactions.

Carbon Monoxide↗

PubMed1979

Dioxygen replacement reaction in myoglobin.

The replacement reaction of myoglobin (Mb), MbCO + O2 leads to MbO2 + CO leads to MbCO + O2, has been studied with flash photolysis in the temperature range from 140 to 320 K and the time range from 2 mus to 200 s. In a fraction of the Mb, the photodissociated CO remains within the protein; rebinding is not affected by the presence of O2 and occurs with rates that are identical with the ones observed earlier in solvents containing only CO. In the remaining fraction CO migrates into the solvent and Mb combines preferentially with oxygen. The rate of the subsequent replacement of O2 by CO permits calculation of the oxygen dissociation rate ko2; ko2 has been determined from 260 to 320 K. The measurements support a multibarrier model.

Animals↗