Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity.

Abstract

A nanosecond absorption spectrometer was used to measure the optical spectra of Hb between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, 2 relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe.sbd.CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.