Resonance Raman spectra are reported for bacterially expressed poplar plastocyanin labeled with S-34, N-15, and Cu-65 isotopes. The strongly enhanced bands near 400 cm(-1) are sensitive to all three isotopes. The strongest bands also exhibit the largest Cu-65, and S-34 shifts, confirming the involvement of Cu-S[cysteine] stretching in the enhancement mechanism. The isotope shift and intensity pattern is similar to that reported recently for P. aeroginosa azurin, but the modes with the largest Cu-S contribution are higher in plastocyanin, 429/420 vs 409 cm(-1). This frequency order is counter to a recently proposed correlation between Cu-S[cysteine] bond strength in Type 1 Cu proteins and the displacement of the Cu from the trigonal [His](2)[Cys] coordination plane toward the distant fourth ligand. The out-of-plane displacement is greater for plastocyanin than for azurin, 0.34 vs 0.04 Angstrom. The small displacement and the weaker Cu-S[cysteine] bond in azurin are attributed to (1) an additional hydrogen bond to the cysteine S atom and (2) an additional donor interaction to the Cu from a backbone carbonyl group. Observed N-15 shifts implicate the N atom of the cysteine peptide, and possibly of other residues, as part of the dynamical unit responsible for the 400-cm(-1) region RR bands. The isotope shifts will provide important constraints on normal mode calculations of Cu protein RR spectra.