Structural observations of time dependent oscillatory behavior of Cu IICl 2 solutions measured via extended X-ray absorption fine structure

Abstract

Cell surface ECTO-NOX proteins exhibit a clock-related, temperature-independent entrainable pattern of periodic (24 min) oscillations in the rate of oxidation of NAD(P)H. Aqueous solutions of copper salts also oxidize NAD(P)H with a similar temperature-independent pattern. For both, five maxima are observed, two of which are separated by 6 min and the remaining three are separated by 4.5 min. In D 2O, the pattern is retained but the period length is proportionately increased to 30 min in direct relationship to the 30 h circadian day observed with D 2O-grown organisms. With copper solutions, periodic changes in redox potential correlate precisely with the periodic changes in the rates of NAD(P)H oxidation. Consequently, the local environment of the Cu 2+ ion in copper chloride solutions was investigated by X-ray absorption spectroscopy. Detailed extended X-ray absorption fine structure (EXAFS) analyses revealed a pattern of oscillations closely resembling those of the copper-catalyzed oxidation of NADH. With CuCl 2 in D 2O, a pattern with a period length of 30 min was observed. The findings suggest a regular pattern of distortion in the axial and/or equatorial oxygen atoms of the coordinated water molecules which correlate with redox potential changes sufficient to oxidize NADH. A metastable equilibrium condition in the ratio of ortho to para nuclear spin orientation of the water associated hydrogen atoms would be kinetically consistent with a 24–30 min timeframe. The temperature independence of the biological clock can thus be understood as the consequence of a physical rather than a chemical basis for the timing events.