Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flowexchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions. ß

The role of the protein environment in determining the redox midpoint potential (E(m)) of Q(A), the primary quinone of bacterial reaction centers, was investigated by mutation of isoleucine at position 265 of the M subunit in Rhodobacter sphaeroides. Isoleucine was changed to threonine, serine, and valine, yielding mutants M265IT, M265IS, and M265IV, respectively. All three mutants, with smaller residues replacing isoleucine, exhibited decreased binding affinities of the Q(A) site for various quinone analogues, consistent with an enlargement or loosening of the headgroup binding domain and a decrease in the van der Waals contact for small quinones. In all other respects, M265IV was like the wild type, but the polar mutants, M265IT and M265IS, had unexpectedly dramatic decreases in the redox midpoint potential of Q(A), resulting in faster rates of P(+)Q(A)(-) charge recombination. For both anthraquinone and native ubiquinone, the in situ E(m) of Q(A) was estimated to be approximately 100 and 85 mV lower in M265IT and M265IS, respectively. The effect on E(m)(Q(A)) indicates destabilization of the semiquinone or stabilization of the quinone. This is suggested to arise from either (i) electrostatic interaction between the partial charges or dipole of the residue hydroxyl group and the charge distribution of quinone and semiquinone states with particular influence near the C4 carbonyl group or (ii) from hydrogen bonding interactions between the hydroxyl oxygen and the N(delta)H of histidine M219, causing a weakening of the hydrogen bond to the C4 carbonyl. The rate of the first electron transfer (k(AB)(()(1)())) in the polar mutants was the same as in the wild type at low pH but decelerated at higher pH with altered pH dependence. The rate of the second electron transfer (k(AB)(()(2)())) was 3-4-fold greater than in the wild type over the whole pH range from 4 to 11, consistent with a larger driving force for electron transfer derived from the lower E(m) of Q(A).

Absorption difference profiles were obtained at wavelengths from 640 to 700 nm with 1-2-ps resolution in a study of primary photoprocesses in the Pr-->Pfr transformation in native oat phytochrome. These experiments were performed using low-intensity laser pulses at high repetition rate; fast sample recycling ensured that essentially all phytochrome species were excited from the Pr ground state. The Pr*-stimulated emission decay at wavelengths > 670 nm exhibits major components with lifetimes of approximately 16 and 50-60 ps. Formation of the asymptotic 695-nm lumi-R absorption spectrum rapidly follows stimulated emission decay. Photoexcitation of one or both of the lumi-R intermediates instantaneously recreates fluorescing Pr* phytochrome, which is spectroscopically and kinetically indistinguishable from that generated by direct illumination of ground-state Pr. This is consistent with assignment of lumi-R as a species in which the chromophore has isomerized from the Z,Z,Z to the Z,Z,E conformation. Anisotropy studies indicate that the orientations of the Pr and lumi-R absorption transition moments are nearly parallel, since little anisotropy decay occurs during the 500-ps time window of these experiments.

Dynamic quenching of the two lifetime component tryptophan fluorescence of Pisum phytochrome has revealed differential accessibility of certain residues. Both acrylamide and Tl+ ions showed preferential exposure of some tryptophans in Pfr-phytochrome. Greater kq's for Pfr are, however, in contrast with values for Avena phytochrome in which Pr-->Pfr conversion impedes Tl+ access. The Pr short lifetime component was more accessible to Cs+; however, the long component accessibility was approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce the total number of fluorescent tryptophans. The absence of the fluorescence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ Ksv's for Pr and Pfr. The two additional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The regions around four of the 10 tryptophans may represent conformationally photoresponsive areas in Pisum phytochrome A. Furthermore, topographic changes associated with the phytochrome phototransformation are not confined to the 58-kDa chromphore domain, and they involve most if not all of the region from Trp-365 to Trp-787. We also provide evidence that the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.

Time-correlated single photon counting was used to observe dynamic quenching of the hypericin and stentorin excited singlet states. The fluorescence quenching data for hypericin and stentorin were interpreted in terms of electron transfer. The observed correlation between free energy change of electron transfer and quenching rate constant suggests that quenching proceeds via electron transfer from hypericin and stentorin to the quenchers. EPR spectra for hypericin, stentorin, and stentorin chromoprotein demonstrated that free radical formation was initiated or enhanced by visible light and that similar radical species were produced in each sample. Furthermore, the EPR signal for stentorin was significantly enhanced by 1,4-benzoquinone, but the overall shape and g-value was unchanged. We suggest that electron transfer in the excited state of these chromophores results in the formation of a cation radical. This electron transfer is a rapid and efficient pathway for deactivation of hypericin and stentorin excited singlet states and should be considered when discussing the photoreactivity of hypericin as a photodynamic agent and of stentorin as the Stentor coeruleus photoreceptor.