Substrate activation by acyl-CoA dehydrogenases: transition-state stabilization and pKs of involved functional groups

The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents-NO2, -CN, and CH3CO-), 3S-C8-, and 3'-dephospho-3S-C8CoA. These analogues lack a beta C-H and cannot undergo alpha,beta-dehydrogenation. Instead they deprotonate at alpha C-H at pH > or = 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as approximately 13.6 (-NO2), approximately 14.5 (-CN), and approximately 14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo alpha C-H deprotonation. While the extent of deprotonation varies, the anionic products from charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3'-dephospho-3S-C8CoA, four pKas at approximately 5, approximately 6, approximately 7.3, and approximately 8 were identified. They were assigned to the following ionizations: (a) pKa approximately 5, ligand (L-H) in the MCADH approximately ligand complex; (b) pKa approximately 6, Glu376-COOH in uncomplexed MCADH; (c) pKa approximately 7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK approximately 8, Glu99-COOH in the MCADH approximately 4NPA-CoA complex. The pKa approximately 6 (b) is not significantly affected in the MCADH approximately 4NPA-CoA complex, but it is increased by > or = 1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The alpha C-H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3'-dephospho-3S-C8CoA in the complex with MCADH are approximately 5, approximately 5, and approximately 6. Compared to those of the free species these pKa values are therefore lowered by 8 to > or = 11 pH units (50 to > or = 65 kJ mol-1) and are close to the pKa of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2'-OH of FAD and Glu376-NH. It is concluded that the pKa shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, alpha C-H abstraction is fast (kapp approximately 55 s-1 at pH 7.5 and 25 degrees C, deuterium isotope effect approximately 1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pKas of approximately 5 and approximately 7.3, which correspond to those determined in static experiments.     

Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis

It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.     

The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.     

Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster of nitrogenase

Substrate reduction by nitrogenase requires electron transfer from a [4Fe-4S] cluster in the iron (Fe) protein component to an FeMo cofactor in the molybdenum-iron (MoFe) protein component in a reaction that is coupled to MgATP hydrolysis and component protein association and dissociation. An [8Fe-7S] (or P-) cluster in the MoFe protein has been proposed as an intermediate electron-transfer site, although how this cluster functions in electron-transfer remains unclear. In the present work, it is demonstrated that one redox couple of the P-cluster (P2+/1+) undergoes coupled electron and proton transfer, whereas a more reduced couple (P1+/N) does not involve a coupled proton transfer. Redox titrations of the MoFe protein P-cluster were performed, and the midpoint potential of the P2+/1+ couple (Em2) was found to be pH dependent, ranging from -224 mV at pH 6.0 to -348 mV at pH 8.5. A plot of Em2 versus the pH for this redox couple was linear and revealed a change of -53 mV/pH unit, indicating a single protonation event associated with reduction. From this plot, it was concluded that p is <6.0 and p is >8.5 in a proton-modified Nernst equation. In contrast, the midpoint potential for the P1+/N couple (Em1) was found to be -290 mV and was invariant over the pH range 6.0-8.5. These results indicate that the protonated species does not influence either the P1+ or the PN oxidation states. In addition, at physiological pH values, electron transfer is coupled to proton transfer for the P2+/1+ couple. The P-clusters are unique among [Fe-S] clusters in that they appear to be ligated to the protein through a serinate-gammaO ligand (betaSer188) and a peptide bond amide-N ligand (alphaCys88), in addition to cysteinate-S ligands. Elimination of the serinate ligand by replacement with a glycine was found to shift the Em values for both P-cluster couples by greater than +60 mV, however the pH dependence of Em2 was unchanged. These results rule out Ser188 as the protonated ligand responsible for the pH dependence of Em2. The implications of these results in understanding the nitrogenase electron-transfer mechanism are discussed.     

Alternative models for describing the acid unfolding of the apomyoglobin folding intermediate

The acid-induced unfolding of the pH 4 intermediate of apomyoglobin (I) is described by either of two models: (1) a Monod-Wyman-Changeux-based model (MWC) where salt bridges perturb the pKa values of specific ionizable side chains, causing unfolding of I as these salt bridges are broken at low pH, and (2) the Linderstrom-Lang smeared charge model (L-L), which attributes acid unfolding of I to charge repulsion caused by the accumulation of positive charge on the surface of the protein. Both models fit earlier acid unfolding data well, but they make differing predictions about the effects of electrostatic mutants, which have been made and tested. Deletions of positive charge within I are found to stabilize I, but disruptions of potential salt bridges have little effect. These results show that the acid unfolding of I (I<-->U) is largely caused by generalized charge effects rather than by the loss of specific salt bridges. Acid unfolding of the native form, which is caused largely by a single histidine with a severely depressed pKa, is a sensitive indicator of changes in stability produced by mutations. In contrast, the I <--> U transition is caused by a number of groups with smaller pKa perturbations and both models predict that the pH midpoint of the I right harpoon over left harpoon U transition is an insensitive indicator of stability. This result reconciles previous conflicting results, in urea and acid unfolding studies of hydrophobic contact mutants, by showing that changes in the stability of I are poorly detected by acid unfolding.     

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pK's of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.     

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.     

Thermodynamics of inositol hexakisphosphate interaction with human oxyhemoglobin

The interaction of inositol hexakisphosphate (IHP) with oxygenated human adult hemoglobin (Hb) was investigated at 25 degreesC. The affinity of IHP for oxygenated Hb is strongly pH-dependent, and potentiometric measurements of proton uptake and release upon IHP addition have shown that over the range between pH 8.0 and pH 6.0 in oxygenated Hb there are three groups of residues that change their pKa values after IHP addition, likely because of their interaction with negative charges of the heterotropic effector. On the basis of previous calculations on the electrostatic properties of human Hb (Matthew, J. B., Hanania, G. I. H., and Gurd, F. R. N. (1979) Biochemistry 18, 1919-1928; Lee, A. W.-m., Karplus, M., Poyart, C., and Bursaux, E. (1988) Biochemistry 27, 1285-1301), two of these groups might be Val1beta and His143beta, which are located in the beta1beta2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropy-driven at pH > 7.     

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.     

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.     

Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM (Ru(NH3)6)3+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b5, respectively. A comparative two-dimensional NMR study of cytochrome b5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 approximately Glu44 segment in cytochrome b5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b5 was studied by optical difference spectra of cytochrome c and variants of cytochrome b5. The association constants (KA) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b5 with cytochrome c, are 4.70(+/-0. 10)x10(6) M-1, 1.88(+/-0.03)x10(6) M-1, 2.70(+/-0.13)x10(6) M-1, and 1.14(+/-0.05)x10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b5 (1.074(+/-0.49)x10(7) M-1 s-1) is higher than those of the mutant protein E44A (8.98(+/-0.20)x10(6) M-1 s-1), E56A (8.76(+/-0. 39)x10(6) M-1 s-1), and E44/56A (8.02(+/-0.38)x10(6) M-1 s-1) at 15 degreesC, pH 7.0, I=0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.     

pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.     

The hydrogen-bonding network of water molecules and the peptide backbone in the region connecting Asp83, Gly120, and Glu113 in bovine rhodopsin

Difference Fourier transform infrared spectra were recorded between mutants of rhodopsin and their batho products. The pigments studied were single and combined mutants of intramembrane residues of bovine rhodopsin: Asp83, Glu113, Gly120, Gly121, and Glu122. Previous studies [Nagata, T., Terakita, A., Kandori, H., Kojima, D., Shichida, Y., and Maeda, A. (1997) Biochemistry 36, 6164-6170] showed that one of the water molecules which undergoes structural changes in this process forms hydrogen bonds with Glu113 and the Schiff base, and that another water molecule is linked to this structure through the peptide backbone. The present results show that this water molecule is located at the place that is affected by the replacements of Asp83 and Gly120 but only slightly by Gly121 and not at all by Glu122. Asp83 and Gly120 are close to each other, in view of the observations that the carboxylic C=O stretching vibration of Asp83 is affected by the G120A replacement and that each replacement affects the common peptide carbonyl groups. Our results suggest that these residues in the middle of helices B and C are linked-through a hydrogen-bonding network composed of water and the peptide backbone-with the region around Glu113.     

Time and pH dependence of the L-to-M transition in the photocycle of bacteriorhodopsin and its correlation with proton release

The pH dependence of the L-to-M transition in the photocycle of bacteriorhodopsin was studied in pump-probe resonance Raman (RR) flow experiments in the range pH 3.5-7.8 on a time scale of 0-700 micros. For pH < 5, following the initial decay of L to M, the two intermediates approach nearly constant levels. From a specially designed perturbation-relaxation experiment at pH 4.6, in which the composition of L and M is perturbed by photoreversal of M, it could be concluded that the incomplete decay of L is due to an intermediate equilibration between L and M. It was found, by both RR and optical transient spectroscopy, that the maximum level of M (approximately 500 micros) increases with pH according to a pKa of 5. 6 (150 mM Na+). Since the proton release from an internal group XH to the extracellular surface is determined by nearly the same pKa of 5.7 [Zimanyi, L., Varo, G., Chang, M., Ni, B., Needleman, R., and Lanyi, J. K. (1992) Biochemistry 31, 8535-8543], it is concluded that this increase is controlled by the dissociation of XH. From the analysis of the perturbation-relaxation experiments and the multiexponential rise of M, a kinetic scheme with two sequential L-M equilibria is proposed for the L-M transition. By comparison with the time behavior of proton release [Heberle, J., and Dencher, N. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5996-6000], it is suggested that it is the second equilibrium which is further shifted toward the M state by the dissociation of XH. From the magnitude of this shift, it is concluded that the L-M transition and proton release are not as strongly coupled as is generally assumed. Instead, it is proposed that structural changes during the photocycle are the dominating factors which reduce the pKa of XH to approximately 5.7 so that proton release becomes possible under normal conditions.     

Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin

The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.     

Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers

Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.     

Axial coordination and reduction potentials of the sixteen hemes in high-molecular-mass cytochrome c from Desulfovibrio vulgaris (Hildenborough)

A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.     

Adhesion molecule expression and endothelial cell activation in cutaneous leukocytoclastic vasculitis. An immunohistologic and clinical study in 42 patients

We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram-negative bacterium Serratia marcescens using finite-difference Poisson-Boltzmann methods for electrostatic calculations and a bead-model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double-stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (-1 e) charge per monomer at pH 7. The most dramatic shift in pKa involves His 89 whose pKa increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments.     

Identification of TaqI endonuclease active site residues by Fe2+-mediated oxidative cleavage

Metal cofactors (Mg2+ and Mn2+) modulate both specific DNA binding and strand cleavage in the TaqI endonuclease (Cao, W., Mayer, A. N., and Barany, F. (1995) Biochemistry 34, 2276-2283). This work attempts to establish the structural basis of TaqI-DNA-metal2+ interactions using an affinity cleavage technique. The protein was cleaved by localized hydroxyl radicals generated by oxidizing Fe2+ within the metal binding sites. Cleavage fragments were separated by SDS-polyacrylamide gel electrophoresis, and cleavage sites were determined using micropeptide sequencing. Eleven amino acid residues in the vicinity of cleavage sites were selected for site-directed mutagenesis. The negative charge at Asp137 is essential for DNA cleavage but not required for sequence specific binding. Mutations at Asp142 abolish both specific binding and catalysis, except for D142E, which converts TaqI into a completely Mn2+-dependent endonuclease. The positive charge at Lys158 appears to be important for both specific binding and catalysis. Mutations at other sites affect binding and/or catalysis to different degrees, except Trp113 and Glu135, which appear to be nonessential for the TaqI enzyme activity. The critical residues for TaqI function are distinct from the PDX14-20(E/D)XK catalytic motif elucidated from other endonucleases.     

Effects of hydrogen bonds on the redox potential and electronic structure of the bacterial primary electron donor

The primary donor, P, of photosynthetic bacterial reaction centers (RCs) is a dimer of excitonically interacting bacteriochlorophyll (BChl) molecules. The two constituents are named PL and PM to designate their close association with the L- and M-subunits, respectively, of the RC protein. A series of site-directed mutants of RCs from Rhodobacter sphaeroides has been constructed in order to model the effects of hydrogen bonding on the redox midpoint potential and electronic structure of P. The leucine residue at position M160 was genetically replaced with eight other amino acid residues capable of donating a hydrogen bond to the C9 keto carbonyl group of the PM BChl a molecule of P. Fourier transform (FT) (pre)resonance Raman spectroscopy with 1064 nm excitation was used to (i) determine the formation and strengths of hydrogen bonds on this latter keto carbonyl group in the reduced, neutral state (PO), and (ii) determine the degree of localization of the positive charge on one of the two constituent BChl molecules of P in its oxidized, radical cation state (P*+). A correlation was observed between the strength of the hydrogen bond and the increase in PO/P*+ redox midpoint potential. This correlation is less pronounced than that observed for another series of RC mutants where hydrogen bonds to the four pi-conjugated carbonyl groups of P were broken or formed uniquely involving histidinyl residues [Mattioli, T. A., Lin, X., Allen, J. P. and Williams, J. C. (1995) Biochemistry 34, 6142-6152], indicating that histidinyl residues are more effective in raising the PO/P*+ redox midpoint potential via hydrogen bond formation than are other hydrogen bond-forming residues. In addition, an increase in positive charge localization is correlated with the strength of the hydrogen bond and with the PO/P*+ redox midpoint potential. This latter correlation was analyzed using an asymmetric bacteriochlorophyll dimer model based on Hückel-type molecular orbitals in order to obtain estimates of certain energetic parameters of the primary donor. Based on this model, the correlation is extrapolated to the case of complete localization of the positive charge on PL and gives a predicted value for the P/P+ redox midpoint potential similar to that experimentally determined for the Rb. sphaeroides HL(M202) heterodimer. The model yields parameters for the highest occupied molecular orbital energies of the two BChl a constituents of P which are typical for the oxidation potential of isolated BChl a in vitro, suggesting that the protein, as compared to many solvents, does not impart atypical redox properties to the BChl a constituents of P.