Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge number of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate experimentally. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiological function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.

Zinc is one of the metal ions essential for life, as it is required for the proper functioning of a large number of proteins. Despite its importance, the annotation of zinc-binding proteins in gene banks or protein domain databases still has significant room for improvement. In the present work, we compiled a list of known zinc-binding protein domains and of known zinc-binding sequence motifs (zinc-binding patterns), and then used them jointly to analyze the proteome of 57 different organisms to obtain an overview of zinc usage by archaeal, bacterial, and eukaryotic organisms. Zinc-binding proteins are an abundant fraction of these proteomes, ranging between 4% and 10%. The number of zinc-binding proteins correlates linearly with the total number of proteins encoded by the genome of an organism, but the proportionality constant of Eukaryota (8.8%) is significantly higher than that observed in Bacteria and Archaea (from 5% to 6%). Most of this enrichment is due to the larger portfolio of regulatory proteins in Eukaryota.

The solution structure of oxidized horse heart cytochrome c was obtained at pH 7.0 in 100 mM phosphate buffer from 2278 NOEs and 241 pseudocontact shift constraints. The final structure was refined through restrained energy minimization. A 35-member family, with RMSD values with respect to the average structure of 0.70 ( 0.11 Å and 1.21 ( 0.14 Å for the backbone and all heavy atoms, respectively, and with an average penalty function of 130 ( 4.0 kJ/mol and 84 ( 3.7 kJ/mol for NOE and pseudocontact shift constraints, respectively (corresponding to a target function of 0.9 Å 2 and 0.2 Å 2 ), was obtained. The solution structure is somewhat different from that recently reported (Qi et al., 1996) and appears to be similar to the X-ray structure of the same oxidation state (Bushnell et al., 1990). A noticeable difference is a rotation of 17 ( 8°of the imidazole plane between solid and solution structure. Detailed and accurate structural determinations are important within the frame of the current debate of the structural rearrangements occurring upon oxidation or reduction. From the obtained magnetic susceptibility tensor a separation of the hyperfine shifts into their contact and pseudocontact contributions is derived and compared to that of the analogous isoenzyme from S. cereVisiae and to previous results.