Search

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

A spectroscopic and functional analysis of two point‐mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild‐type BR, the M formation in BR Asp85–‐Glu is accwelerated approximately 10‐fold, whereas the M decay in BR Asp96–‐Asn is slowed down approximately 50‐fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85–‐Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96–‐Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

The transport activity of purified Na+K+‐ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+‐ATPase from pig kidney were attached to planar lipid bilayers in a sandwich‐like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3‐1‐(2‐nitro)phenylethyladenosine 5′‐triphosphate, caged ATP] ATP was released after illumination with u.v.‐light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+‐ATPase‐specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+‐ATPase with a large variety of different parameters.

Purified Na+,K+‐ATPase from pig kidney was attached to black lipid membranes. Pump currents of the enzyme could be measured with a time resolution of approx. 1 ms by releasing ATP from caged ATP with a UV laser flash. Analysis of the transient currents shows that a slow non‐electrogenic step is followed by an electrogenic transition with a rate constant of 100 s−1(22°C). The exponential components found in the transient currents are compared to transitions in the Albers‐Post scheme.

Purified Na +,K +-ATPase from pig kidney was attached to black lipid membranes. Pump currents of the enzyme could be measured with a time resolution of approx. 1 ms by releasing ATP from caged ATP with a UV laser flash. Analysis of the transient currents shows that a slow non-electrogenic step is followed by an electrogenic transition with a rate constant of 100 s −1(22°C). The exponential components found in the transient currents are compared to transitions in the Albers-Post scheme.

The magnetostatic properties of K2CoxFe1-xF4 have been investigated by susceptibility and Mössbauer measurements at different concentrations and temperatures. Three magnetically ordered phases are found: a planar phase, an OAF phase, and an uniaxial phase. The experimental results are compared to a molecular-field calculation. At certain concentrations and temperatures it is not possible to obtain a good least squares fit of the Mössbauer spectra, which is tentatively assigned to relaxation effects due to fast spin fluctuations at the OAF phase transition.