Paramagnetic Cobalt and Nickel Derivatives of Alcaligenes denitrificans Azurin and Its M121Q Mutant. A 1 H NMR Study † Jesu ´s Salgado, Hermas R. Jime ´nez, and Jose ´ M. Moratal* Department of Inorganic Chemistry, UniVersity of Valencia, Dr. Moliner 50, 46100 Burjassot (Valencia), Spain Sandra Kroes, Gertrud C. M. Warmerdam, and Gerard W. Canters Gorlaeus Laboratories, UniVersity of Leiden, Einsteinweg 55, 2300 RA Leiden, The Netherlands ReceiVed July 28, 1995; ReVised Manuscript ReceiVed December 6, 1995 X ABSTRACT: Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1 H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gln in stellacyanin. Although its physiological function is still unclear (it is presumably part of the denitrification electron transfer chain in denitrifying bacteria), azurin continues to be one of the favorite subjects for the study of biological electron transfer (Adman, 1985; Sykes, 1991; Chapman, 1991; Canters & van de Kamp, 1992; Wuttke & Gray, 1993). Many reasons contribute to this. First, it is one of the best known electron transfer proteins, and its structural, chemical, and spectro- scopic features have been investigated by almost all the suitable physical techniques (Chapman, 1991). Furthermore, since its gene was cloned and expressed in Escherichia coli (Canters, 1987; Arvidsson et al., 1989), many different site- directed mutants have been obtained and studied, allowing the understanding of the particular relevance of almost all of the potentially important residues for the functional, structural, and spectroscopic properties of the protein (van de Kamp et al., 1990a, 1993; Hoitink et al., 1992; van Pouderoyen et al., 1994; Pascher et al., 1993; Nar et al., 1991a; Romero et al., 1993; den Blaauwen & Canters, 1993; Mizoguchi et al., 1992; Germanas et al., 1993; Canters & Gilardi, 1993). The crystal structures of azurins from different sources and in the two possible redox states of the copper ion, in the apo-protein as well as in various azurin metalloderivatives and site-directed mutants, have been solved in the last several years (Adman & Jensen, 1981; Nar et al., 1991a,b, 1992a,b; Romero et al., 1993; Baker, 1988; Shepard et al., 1990, 1993; Moratal et al., 1995; Blackwell et al., 1994). In the wild type copper protein, the metal ion is strongly bound to the Sγ of Cys112 and to the Nδ of both His46 and His117 (Baker, 1988; Nar et al., 1991b). This basic equatorial trigonal planar entity is claimed to be the essential part of the type 1 copper site, although mutagenesis studies of His46 (Germanas et al., 1993), His117 (den Blaauwen & Canters, 1993), and Cys112 (Mizoguchi et al., 1992) have demon- strated that Cys112 is the only coordinated residue which is absolutely essential for a blue copper center. The Sδ of Met121 and the carbonyl oxygen of Gly45 are weakly ligated, resulting in a distorted trigonal-bipyramidal geom- etry (Figure 1A) (Baker, 1988; Nar et al., 1991b). Although the role of these axial ligands is not completely understood, it is thought that they are responsible for modulating the redox potential of blue copper proteins (Gray & Malmstro ¨m, 1983). Thus, azurins are unique in having a carbonyl glycine in the coordination sphere. Other blue copper proteins like stellacyanin, cucumber peelings cupredoxin, and umecyanin probably present an oxygen, from a glutamine side chain, in the axial position instead of the methionine sulfur which is common to the rest of the type 1 copper protein family (van Driessche et al., 1995). Metal substitution has been frequently used for the structural and spectroscopic characterization of type 1 metal sites by different techniques (Nar et al., 1992a; Moratal et al., 1993a-c, 1995; Blackwell et al., 1994; McMillin et al., 1974; Tennent & McMillin, 1979; Strong et al., 1994; Di Bilio et al., 1992; Germanas et al., 1993; Hill et al., 1976; Blaszak et al., 1982; Dahlin et al., 1989; Piccioli et al., 1995; Salgado et al., 1995; Vila, 1994; Danielsen et al., 1995). Zinc, nickel, and cadmium azurins have been crystallized and their structures solved by X-ray diffraction (Nar et al., 1992a; † This work was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO), by the Spanish “Direccio ´n General de Investigacio ´n Cientı ´fica y Te ´cnica”, (DGICYT, PB 91- 0639), and by a fellowship from the Autonomous Government of Valencia (J.S.). * Author to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, January 15, 1996. 1810 Biochemistry 1996, 35, 1810-1819 0006-2960/96/0435-1810$12.00/0 © 1996 American Chemical Society