Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H(+) release, an important step in the normal light-induced H(+) transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H(+)-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H(+) release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.

In 1994, Fuji Photo Film Co., Ltd., Japan, filed a patent for nonaqueous Li-ion batteries in which tin composite oxides (TCOs) were used as the active anode material. 1 Since then, much attention has been given to the potential of substituting this class of compounds for carbon negative electrodes in Li-ion batteries. Tin oxide composites are attractive because of their high gravimetric and volumetric specific capacities relative to carbon, which enable cells of higher energy densities to be built. 2 It is reported 2 that most tin composite oxides are amorphous and the Sn(II)-O sites are active for lithium insertion. Although some early Li 7 NMR measurements have been rationalised in terms of an intercalation mechanism for charge and discharge reactions, more recent experimental results based on X-ray diffraction (XRD) analyses of the active materials at different stages of charge and discharge are more satisfactorily explained by the alloying mechanism made famous by Dahn and associates. 3,4 The alloying mechanism assumes the formation of a lithia (Li 2 O) matrix mostly during the first charging cycle. This matrix binds the lithium-tin regions together and smoothens the large volume changes associated with alloying and dealloying in reversible charge and discharge reactions.Both pristine and doped tin oxides 2-6 have been investigated as anode materials. All of these materials show reversible capacities higher than 500 mAh/g but large capacity losses in their first cycles. They were synthesized by classical solid state reactions involving two or more components. In this study, we consider amorphous and crystalline Sn 2 P 2 O 7 as intrinsic P-doped tin oxide composites, and investigated their performance as anode materials for Li-ion batteries. We have chosen Sn 2 P 2 O 7 to circumvent the mixing problem inherent in most solid state reactions. We are particularly interested in the effects of crystallinity on the electrochemical performance of Sn 2 P 2 O 7 . ExperimentalMaterials preparation.-Crystalline Sn 2 P 2 O 7 (98%, Aldrich) was used as received. For the preparation of amorphous Sn 2 P 2 O 7 , crystalline Sn 2 P 2 O 7 was pelletized at 2 ϫ 10 6 Pa and fired in flowing nitrogen in a Lindberg/Blue M tube furnace. After 2 h at 900ЊC, the melt was removed from the furnace and immediately quenched in air between two stainless plates at room temperature. The solid thus formed was carefully ground to powder and the above procedure repeated once more using the ground Sn 2 P 2 O 7 powder as the starting material, and eight more hours of heating at 900ЊC.Composition and structure determinations.-Elemental composition was determined by inductively coupled plasma (ICP) spectroscopy on a Perkin Elmer Optima 3000DV, using digestions of the melt-quenched samples in 1:1 (by volume) mixture of HCl/HNO 3 at 60ЊC. The crystal structure of Sn 2 P 2 O 7 was confirmed by XRD, using a Philips PW1710 diffractometer and Cu K␣ X-ray source ( ϭ 1.54 Å). The 2 range was initially set at 10-70Њ, but was subsequently narrowed to 10-42Њ ...

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.

Natural-abundance 15N NMR spectroscopy on dodecylguanidine reveals solvent and protonation effects that model those that could occur for the arginine side chain in proteins. Our results demonstrate that the 15N chemical shifts of the terminal guanine nitrogens strongly depend on the solvent chosen for measurements. A polar H-bond-donating solvent like water has strongly deshielding effects on the neutral guanidine group (with the latter acting predominantly as an H-bond acceptor). As a result, a substantial upfield shift occurs when neutral guanidine is dissolved instead in a non-H-bonding solvent (chloroform). These solvent effects can be as large as those induced by protonation changes. This limits the ability of 15N chemical shifts to distinguish the protonation state of the arginine side chain, at least without specific knowledge of its environment. These results help to reconcile previous interpretations about the protonation state arg-82 in the M state of bacteriorhodopsin based on FTIR and 15N NMR spectroscopy. That is, contrary to earlier conclusions from solid-state NMR, the side chain of arg-82 could undergo a deprotonation between the bR and M states, but only if it also experienced a significant decrease in the H-bonding character and polarity of its environment. In fact, the average 15N chemical shift of the two Neta of arg-82 in bacteriorhodopsin's M intermediate (from the previous NMR measurements) is 17 ppm upfield from the corresponding value for the deprotonated arginine side chain in aqueous solution at pH >14, but only 3 ppm upfield from the value for deprotonated dodecylguanidine in chloroform.