The Escherichia coli protein RecA catalyzes DNA strand exchange necessary for DNA repair and homologous genetic recombination. The binding of adenosine triphosphate (ATP) to RecA induces a conformation with a high affinity for DNA. However, adenosine diphosphate (ADP) binding results in a RecA conformation with a lower affinity for DNA. In an attempt to identify and compare the specific structural changes induced by the binding of the two nucleotides, we have utilized Fourier transform infrared difference spectroscopy in conjunction with the photolytic release of caged nucleotides. RecA-ADP minus RecA and RecA-ATP minus RecA difference spectra contain distinct, reproducible changes in the 1800-1300 cm(-1) region associated with nucleotide binding to RecA. In addition, the first difference FT-TR evidence suggests that small structural perturbations in the RecA and/or nucleotide are responsible for differences between the low and high DNA affinity states of the protein. The results presented here are consistent with studies that predict the involvement of key amino acid side chains and also reveal secondary structural changes may be associated with nucleotide binding to RecA in the absence of DNA.