Metal Replaced Hemoproteins L. Charles Dickinson University of Massachusetts Amherst. 01002
A review with introductory laboratory preparation of cobaltmyoglobin
I t is clear that great progress is being made in the molecular level understanding of many of the fascinating processes of living organisms. Much of this progress has come through separation and examination of components by application and extension of modern chemistry. Among the exciting areas of comparatively recent development are the coding of protein amino acid sequences by the DNA polynucleotides, the structure and function studies of cell membranes with the very interesting transport of ions through the phospholipid hilayer, the extensive detail of many enzyme structures and the chemistry of their highly selective mechanisms as revealed in atomic detail through X-ray crystallography, magnetic resonance, or impressive chemical modification of individual residues, bioenergetics of muscle tissue, etc., etc. Initially, much of the chemical work on enzymes depended upon natural variations among mutants or species. More recently, chemical modification of specific residues has contributed much to knowledge of binding sites and mechanisms. Another method of chemical modification in those cases where metal ions play a crucial role in the function of the molecule is to replace the metal. For metal activated enzymes such as carboxypeptidases, enolases, or phosphatases, the native metal may be removed by dialysis or chemical chelating agents, and a different metal added. This paper deals with a different class of metal-containing enzymes, hemoproteins, where metal replacement requires rather more chemistry related to the metal substitution in porphyrin rings. As detailed below, these metal replaced enzymes have led to increased structural detail in bonding of substrates to metals-oxygen to cohalthemoglohin, peroxide to manganese peroxidases, and the role of specific metal and metal-ligand bonds in determining the affinity of hemoglobin for oxygen and the ahility of cytochrome c to transfer electrons. Even though these bio-molecular problems are the focus of excitement for many active chemists, it seems many chemists in education a t the crucial formative undergraduate stage of their students' choice of major career direction shy away from these systems or a t best treat them so trivially that one does not taste the fascination. The rea-
sons for this are many, ranging from cynicism of their own or their students' comprehension which may come out as "these things are too complex or too messy" to what appears to be a sort of neo-vitalistic expression of the same thing "you can't fool around with those molecules in uitro and expect them to behave like they do in the organism.'' This paper is an attempt to dispel some of this reluctance to get involved in biologically important molecules. In particular, a review of some basics and some recent work on metal replacement in hemoproteins is given to show how this work has increased our knowledge of the structure and function of the native hemoproteins, and, hopefully, transfer some sense of excitement and interest in this developing field. As an appendix, a preparation of cobalt myoglobin developed and tested in the author's lahoratory is described in sufficient detail to be accomplished by a motivated upperclass undergraduate. Hemes and HemoproteinChemistry
Proteins are, of course, sequences of a-amino acids of varying length joined together by eliminating water and forming a peptide bond between the carboxyl carbon of one amino acid and the nitrogen of the a-carbon of another. aAmino acids have a great variety of side chains and thus can play different roles of hydrophobicity, cross-linking, helix formation, polarity, etc. within the protein molecule. Most proteins are colorless, hut some have a smaller molecule attached to them which makes them red or brown and gives them ability to do a rather startling variety of special chemistry with small molecules such as oxygen or hydrogen peroxide or to serve as electron receivers or senders. This colored molecule is the heme and its structure is shown in Figure 1. The metal free ring structure is called protoporphyrin IX (PPIX) and is purple. The red color in ferrous heme arises from an absorption band a t 550 nm in the visible region. A near ultraviolet peak a t about 410 nm is called the Soret band, has approximately ten times the extinction coefficient of the visible hand, and is characteristic of porphyrin macrocycles. The Soret and visible bands shift with
Summarv of Results for Metal Redaced Hemooroteins Hemoprotein Hernodobin (human, horre)
Metal
Function Preserved
CO
i) Reversible oxygenation
CU, Mn, A9
None
Function Valuen
P I A ~= 105 i5)mm
Reference Illbl
li) Cooperativity C r R , , Rh
Myoglobin (sperm whale) cytochrome c (horre heart) CytoChrome c Peroxidare (baker's yeast)
Reversible oxygenation
c",
None
Mn. Cu. AP. N i
i) + I l / + l i l oxidation State6 BCCeSSibie ii) oxidare activity undetemined
Mn. Ag cr. Ru. Rh CO
Mn
catalytic peroxide reduction
Qvalue in oarentherir i s for native iron hemoorotein.
f v ,,I
