Previous C13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pKa of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pKa = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.

however, the transport rate decreases by a factor of -2 in bRHFr compared with bRHCI'. The data indicate that in the acid purple form bR transports the halide anions instead of protons. We present a testable model for the transport mechanism, which should also be applicable to halorhodopsin.Halobacterium halobium contains two light energytransducing pigments: bacteriorhodopsin, with an absorption maximum at 568 nm (bR5M), which transports H+ out of the cell; and halorhodopsin (hR), which transports Cl-into the cell. In the cell membrane and in the purified state, bR occurs in the form of small membrane patches, the purple membrane, which are two-dimensional crystals ofbR and lipid (for review, see refs. 1-4). Both pigments are intrinsic membrane proteins, contain nearly identical all-trans-retinylidene chromophores, and show extensive sequence homologies (for review, see ref. 5).The absorption maximum of bR is red-shifted from 568 to 605 nm at low pH, forming blue bR, or bRF d, and transport activity ceases (6-8). The same effect is obtained by removing residual metal cations with an ion-exchange resin because the high negative surface charge of the purple membrane then drops the surface pH below 1.0 (9, 10). The addition of Clreverses the red shift and restores charge transport in this "acid purple" form (bR5HC) (11). The absorption changes accompanying charge transport ofbR568 are changed in bR5'and resemble those of hR (12). The question arises, which ion is translocated by the bR54 form?Direct measurements of the expected changes in Cl-or H+ concentration under the required low pH conditions are difficult and so far have not succeeded. However, substantial information on the ion transport in bR and hR has been obtained by measuring the kinetics of the absorption changes and charge shifts that accompany ion transport, and these should be sensitive to the properties of the transported ion. We have, therefore, measured current and absorption kinetics ofpurple membrane after substituting 2H' for H+ and Brfor Cl-. Techniques and results of photoelectric measurements have recently been reviewed comprehensively (13) and will not be described here. MATERIALS AND METHODSThe bR-containing purple membrane fragments were prepared from H. halobium strain JW-3 (ET 1001) according to the standard procedure (14). After orientation in an electric field, they were immobilized in a polyacrylamide gel (15), and slabs of 10 x 5 x 2 mm were placed in a cuvette. The gel samples were preequilibrated in the desired solution overnight, and the pH was monitored with an OP-0808 electrode (Radelkis, Budapest) and adjusted by adding appropriate amounts ofconcentrated HCI or HBr solutions (Aldrich). The measured p2H values were corrected by subtracting 0.41 pH unit.Optical and electrical signals were measured after laser pulse excitation at 532 nm from a frequency-doubled neodymium-yttrium/aluminum garnet (Nd-YAG) laser with 8-ns pulse length and 1.5-mJ pulse energy. To record photosteadystate currents, we used 0.8-to 4-s-long illuminatio...

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CObound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as ''kinetic hole burning.'' The study of these correlations gives direct insights into the structure-function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates inf luence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.