Mechanism of Redox- and CO-Sensing by the Heme Protein, CooA: Cryoradiolysis Studies
Gas-sensing heme proteins participate in a variety of signal-transduction mechanisms across a broad range of biological systems. This class of proteins functions by undergoing an allosteric conformational change in response to the binding of a small gas molecule to a heme group. CooA, which is a redox- and CO-sensing transcription factor that is found in several bacteria, regulates gene expression that enables growth on CO as a sole energy source. In the current study, we have employed the technique of gamma-irradiation / cryoreduction spectroscopy (cryoradiolysis) to investigate facile changes that occur to the heme coordination structure of CooA during activation. Specifically, we conducted cryoradiolysis experiments to elucidate the chemical steps of an unusual ligand switch that occurs rapidly when the CooA hemes are reduced from the Fe(III) to Fe(II) state. Presented are our protocols for the preparation of samples suitable for cryoradiolysis experiments, as well as results we have obtained that provide evidence for the isolation of a highly-reactive Fe(II)-Cys/Pro species upon gamma irradiation at 77 K. Finally, we present initial results of annealing experiments that seek to identify the chemical details of the redox-mediated ligand switching mechanism utilized by CooA from Rhodospirillum rubrum.
Clark, Robert W. and Kalafut, Deanna F., "Mechanism of Redox- and CO-Sensing by the Heme Protein, CooA: Cryoradiolysis Studies" (2008). Chemistry Faculty Publications. Paper 9.