NUCLEAR MAGNETIC RESONANCE STUDIES OF THE "BLUE COPPER" PROTEIN PLASTOCYANIN (TWO-DIMENSIONAL NMR, ELECTRON TRANSPORT, PHOTOSYNTHESIS, PROTEIN STRUCTURE)

CHRISTOPHER LEN KOJIRO, Purdue University

Abstract

Plastocyanins (pcy) from a higher plant spinach, Spinacia oleracea , a cyanobacterium Anabaena variabilis , and a green alga Chlorella fusca , were studied by ('1)H and ('13)C NMR. Two-dimensional (2D) heteronuclear ('13)C ('1)H chemical shift correlated (HETCOR) results led to correction (reversal) of the previous assignments (Markley et al. (1975) Biochemistry 14, 4428) of the C(,(delta))-('1)H and C(,(epsilon))-('1)H peaks of histidine-37 in A. variabilis pcy. 2D nuclear Overhauser effect spectra showed a similar reversal of the His-37 peaks with spinach pcy. ('13)C ('1)H HETCOR results showed the Tyr-83 C(,(delta))-('1)H and C(,(epsilon))-('1)H chemical shifts published for higher plant pcys (Cookson et al. (1980) Biochim. Biophys. Acta 591, 162) are reversed in A. variabilis pcy. Peaks from the two copper ligated histidines in A. variabilis pcy showed pH dependent ('1)H chemical shifts similar to those of spinach pcy: His-87, pK(,a)'= 5.1. The pH dependence of the His-37 peaks was shown to be spectroscopic shifts which correlate with the titration of His-87. ('1)H peaks assigned to methyl groups of spinach and A. variabilis pcys exhibited pH dependent chemical shifts, which correlate with the titration of His-87 and indicate parallel structural changes of non-metal ligand residues. Line widths of the ring ('1)H peaks of His-87 are field dependent, allowing calculation of the reduced lifetime ((tau)(,red)) for a proton on N(,(delta)) in 20 mM phosphate buffer (PO(,4)) : A. variabilis pcy, (tau)(,red) (DBLTURN) 400 (mu)sec; spinach pcy, (tau)(,red) (GREATERTHEQ) 946 (mu)sec. The exchange rate of this proton increases as the PO(,4) concentration increases. A model explaining the exchange broadening in terms of general base catalysis by the PO(,4) was derived and shown to agree with the observed results, although a PO(,4) dependent electrostatic or conformational effect could not be excluded. All three histidine ('13)C(,(gamma)) peaks in A. variabilis pcy were assigned. Previous assignments of the homologous ('13)C(,(gamma)) peaks in spinach pcy (Markley et al. (1977) Biochem. Biophys. Res. Commun. 78, 106) were corrected. A glutamic acid ('13)C(,(delta)) peak was found to titrate with an unusually high pK(,a)'((TURN) 5.3); this peak was assigned to the N-terminal Glu-(-2). Exchange of ('113)Cd(II) for Cu(I) led to changes in chemical shifts of groups at the metal binding site. Spectral changes observed with residues as far as 18 (ANGSTROM) from the metal site indicate that the structure at the metal binding site is coupled to the structure at the opposite end of the molecule.

Degree

Ph.D.

Subject Area

Biochemistry

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