Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP). The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF). We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1. The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil. CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS. In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS. Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy. The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths. Removal of metal ions produces more poorly dispersed peaks with sharper line widths. Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths. Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex.