THE METHOD OF TIME-RESOLVED SPIN-PROBE OXIMETRY - ITS APPLICATION TO OXYGEN-CONSUMPTION BY CYTOCHROME-C-OXIDASE

This work broadens the scope and improves the time resolution of spin-probe oximetry, a technique in which small nitroxide spin probes detect oxygen consumption via change in their relaxation properties [Froncisz, W., Lai, C.-S., & Hyde, J. S. (I 985) Proc. Natl. Acad. Sci. U.S.A. 82, 411-415]. For rapid oxygen kinetic studies we combined the methodology of spin-probe oximetry with a recently developed loop-gap resonator, stopped-flow EPR system [Hubbell, W. L., Froncisz, W., & Hyde, J. S. (I 98 7) Rev. Sci. Instrum. 58, 1879-18861. The technique used microliter volumes of reactant solutions. Enzymatic consumption of oxygen by cytochrome c oxidase in the presence of ferrocytochrome c substrate was followed continuously in time under limited-turnover conditions, where the concentration of oxygen consumed often was comparable to or less than the amount of enzyme present. In detecting less than micromolar oxygen concentration changes, we have achieved a time resolution of the order 30 ms when flow is stopped. Oxygen consumption was followed under two different limited-turnover conditions: In the first, the amount of oxygen consumed was limited by available ferrocytochrome c, and the time course of oxygen consumption and its pH dependence were compared with the optically detected ferrocytochrome c consumption. In the second, the oxygen consumed was ultimately limited by the availability of oxygen itself while ferrocytochrome c was regenerated and remained in excess. The overall amount of oxygen consumed was of the order of 10 times the amount of enzyme, and more rapid oxygen consumption was observed for enzyme in the prereduced "pulsed' form [Antonini, E., Brunori, M., Greenwood, C., Colosimo, A., & Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-31321 than in the 'resting" form. These latter oxygen-limited experiments showed a zeroth-order rate dependence on oxygen concentration down to an oxygen concentration of less than 1-mu-M. Below 1-mu-M oxygen, where oxygen itself became rate limiting, our technique enabled us to observe the oxygen consumption rate diminish to zero in a period of less than 1 s and to estimate the oxygen concentration where the rate diminished to half-maximal value.