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DISCLAIMER: What you see below is a rough draft of a tutorial section. I am in the process of rewriting it and making the figures. Kyle and I updated it on Dec. 31, 2004.

Please come back in a bit (or quite a bit!) and see what else has developed and improved. --Jim

 

INTRODUCTION TO HEMES AND HEME PROTEINS

 

Here at the Hemeteam we study the interrelationship between structure, function and molecular dynamical properties of heme proteins and heme enzymes. Research emphases within our research group vary with the interests and abilities of students, postdocs and visiting faculty. At any time one may find projects ranging from Biophysical Chemistry to Protein Engineering. The following description serves to introduce the site visitor to hemes and heme proteins in general. A separate section, "Current Research", describes specific proteins currently under study. For further depth you can go to the "Current Research Results" section of this site for presentation of current papers and manuscripts.

Contents:

A) The Heme Moiety

B) First Glimpse of Heme Protein Structures

C) B-Type and C-Type Heme Proteins

D) Heme Binding (Coordination) to Proteins

E) Active Sites and Natural Heme Coordination Structures

F) Active Site Chemistry Occurs at the Heme

G) Heme Iron Ion Oxidation States--The Key to Redox Proteins

H) Heme Iron Ion Ligation--The Key to Oxygen Regulation

I) Heme Ligation is a General Process

J) Magnetic Properties, Coordination Number and Structure

 

A) The Heme Moiety

All heme proteins contain a tetrapyrrole macrocycle, such as protoporphyrin IX, shown in Fig. 1. Structures such as these are termed porphyrins and these macrocycles are essentially planar structures. Nature, and Organic Chemists, have provided us an immense array of distinct porphyrins that differ in the number and arrangement of their peripheral substituents. For example, the porphyrin deuteroporphyrin IX is derived from the protoheme IX structure but with the peripheral vinyl groups replaced by -H.

A porphyrin macrocycle can bind a single metal ion in its central "hole" by four N-Fe bonds (Fig. 1). Each N-Fe bond is a "coordinate-covalent" bond (see also Sec. D) and the overall number of bonds is called the coordination number (mono-, di-, tri-, tetra-, penta-, hexa- coordinate, etc). The resulting metallomacrocycle is more specifically called a metalloporphyrin or a heme. Throughout this narrative "heme" will always refer to an Iron-porphyrin.

To further introduce common terminology we note that in general, anything that forms a bond with a metal ion is called a "ligand", and the structure of the resulting complex may be termed the "ligation structure" or "coordination structure". Hence, porphyrins are ligands.

Further, if a single ligand can provide more than one bond to a metal ion it is called a chelate and it is further classified by the number of bonds it can make with the metal ion. Bidentate = 2 bonds, tridentate = 3 bonds, tetradentate = 4 bonds, hexadentate = 6 bonds, etc. So, porphyrins are tetradentate chelate ligands for iron ions!

Fig. 1

 

There you have it: A brief tour of Metal Coordination Chemistry...which you might remember from General Chemistry and/or Inorganic Chemistry courses! Now let's look at two examples of important heme proteins.

Heme-b (protoporphyrin IX; Fig. 1) in its iron-bound form (Hemin; Fig. 1) is the active site prosthetic group for a number of proteins that are widely distributed in nature. This includes vertebrate, invertebrate, and plant Hemoglobins (Hbs) and Myoglobins (Mbs), heme-peroxidases, cytochrome P450s, heme-oxygen sensors, heme CO-sensors, heme-NO sensors and heme-redox sensors, to name just a few. Reiterating: protoporphyrin IX is the tetradentate chelate "ligand", coordinating the central iron ion by four N-Fe coordinate covalent bonds. Consistent with the porphyrin structure noted earlier, the average structure of Heme-b outside a protein matrix can be thought of as reflecting "planar ligation" of the iron ion by protoporphyrin IX. However inside proteins heme's may deviate significantly from planarity.

Another example of such a naturally occurring iron-ligand complex is Heme-a (Fig. 2), one of the two hemes (Heme-b is the other) found in the multi-metal enzyme Cytochrome c Oxidase (CCO). Whereas its core structure is similar Heme-b, it has a very long and perhaps "floppy", unsaturated peripheral side chain that distinguishes it.

It is the metal-bound forms of porphyins (and related structures called corrins, phlorins, etc) that serve as the biologically functional active-site prosthetic groups for many proteins, because metals imbue these sites with multifaceted chemistry not otherwise achievable.

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B) First Glimpse of Heme Proteins

 

In heme proteins the heme groups are embedded into the three dimensional structure formed by the protein's polypeptide chain, as shown by the structure of the myoglobin-like oxygen binding protein GMH4 monomer hemoglobin whose NMR structure we determined and is shown in Fig. 3. Only in this combination of heme + polypeptide chain, called the holoprotein or holoenzyme, are heme proteins properly functional. Total protein function is due to both heme and protein (polypeptide) properties.

Fig. 3

Average NMR Structure of G. dibranchiata monomer component 4 hemoglobin

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C) B-Type and C-Type Heme Protein Structures

 

B-type hemes such as those shown in Figs. 1 and 2 have a maximum coordination capacity of six ligating atoms. The heme itself accounts for four of these position arranged approximately in a plane. Heme-bs are held in proteins by a single axial coordinate-covalent bond from an amino acid side chain to the heme iron, as shown in Fig. 4, and by the hydrophobic effect provided by the structure and amino acid side chains of the heme pocket (Figs. 3-6). Because there are no other attachments to the protein by the heme, except for the axial ligation, heme-b.can be easily extracted from b-type heme proteins. Once the heme is removed the remaining protein polypeptide, which is typically partly unfolded and non-functional, is called the apoprotein. In b-type heme proteins the last vacant coordination position, also axial is free to be used for ligand binding and/or catalysis.

Fig. 4

Fig. 5

Oxy-DosH X-Ray Crystal Structure

Fig. 6

X-Ray Crystal Structures of Oxy-DosH (A) and Deoxy-DosH

While c-type heme proteins also have a heme imbedded in their three-dimensional protein structure (ie cytc; Fig. 7), they possess a unique heme that cannot be readily extracted because it is covalently attached to the polypeptide chain at two points: through the 2- and 4- heme positions.. In nature this covalent attachment comes about as a result of a biocatalyzed reaction between two heme peripheral vinyl groups (see heme b, Fig. 1) and two cysteine amino acid side chains, to form two separate thio-ether linkages, as shown in Fig. 8. In nature the protein polypeptide and heme are synthesized by separate pathways. In contrast to heme-b proteins the heme-c proteins are said to have covalently bound hemes.

Fig. 7

X-ray Crystal Structure of Horse Cytrocome c

Fig. 8

Cytrocome c Heme Ligation and Covalent Attachments

to Cys14 and Cys17

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D) Heme Binding (Coordination) to Proteins

 

Porphyrins coordinate (bind) a variety of metal ions, but iron is by far the most common metal ion in physiological heme proteins. Removable (ie non-covalently bound) hemes are the most frequently encountered in nature. However, hemes also bind to proteins via the metal ion, independent of whether a specific heme type is covalently bound to the polypeptide.

Reviewing the contents of Sec. A, this type of bond is a coordinate-covalent bond (which you should have learned about in General Chemistry or Inorganic Chemistry!).

1. A ligand binding to a metal ion (a process known as ligation or coordination) results in formation of a coordinate-covalent bond between the metal and ligand. Example: a porphyrin binding an iron ion.

2. ALSO: In the case of the heme proteins that we study ligation also occurs when a heteroatom from amino acid side-chain of the polypeptide acts as ligand, and in this case as a Lewis Base by donating two electrons to "coordinate" the iron (which acts as a Lewis Acid):

L: + Fe(II) --> L:Fe(II).

3. In heme proteins this attachment of the heme to the polypeptide is a labile bond, which accounts for the ease of removal of b-type hemes, noted in Sec. C. In fact, chelation or binding of the iron ion by heme-b, as shown in Fig. 1 is due to the heme acting as a "tetradentate" ligand, providing four coordination capacity from the four pyrrole nitrogens in the macrocycle.

Clearly, only amino acids that contain heteroatoms in their side-chains may coordinate hemes. This limits the candidate list to: Arg, Lys , His, Thr, Ser, Met, Trp.

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E) Active Sites and Natural Heme Coordination Structures

 

The following group of schematic structures shows some of the common protein-supplied coordination structures found in heme proteins. In each case the heme group is drawn as an atom-skeleton consisting of dark circles. The list of proteins that display each coordination type is not complete.

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F) Active Site Chemistry Occurs at the Heme

 

The Heme group is what makes a heme protein special, by virtue of the combined chemical, magnetic and spectroscopic properties of the aromatic porphyrin and the transition metal ion. It is the site of function and the locus of heme protein activity.

To fully appreciate how heme proteins function it is necessary to understand some basic transition metal chemistry, since Fe is a first row transition element. Particularly important is the interrelationship between heme iron ion oxidation state, coordination number and structure.

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G) Heme Iron Ion Oxidation States--The Key to Redox Proteins

Transition metals commonly occur in more than one oxidation state. The common oxidation states for iron are +2 (ferrous) and +3 (ferric), with the +4 state less common, but still found both in nature and in the laboratory. Figure 3 lists the common oxidation states for many heme proteins.

Several heme proteins (cytc, peroxidases, P450, NOS) access more than one of these iron oxidation states in performing their function. The ability to support such redox chemistry and electron transfer is key to the variety of chemistry that makes heme proteins so interesting.

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H) Heme Iron Ion Ligation--The Key to Oxygen Regulation

 

As noted in Section A, the terms "ligation" and "coordination" describe the number and types of bonds to the heme iron ion. The three oxidation states (+2, +3, +4) of Fe can accomodate at most "hexadentate" coordination, meaning that iron ions may make only a total of six coordinate-covalent bonds. The ligands of such a "coordinatively saturated" Fe ion must be arranged in an octahedral coordination geometry (or ligation structure), or something closely approximating octahedral geometry.

Reviewing what we know (refer to Fig. 4.)

1. Maximal Coordination Number for an Iron ion is 6.

2. A porphyrin provides 4-coordination (Sec. A) in a planar geometry (Equitorial ligation in Fig. 4).

3. An amino acid from the polypeptide provides 1-coordination (Sec. D) (Axial ligation in Fig. 4).

4. That leaves 1 coordination place vacant--An Axial ligation site in Fig. 4.

 

 

Fig. 4 Heme coordination with axial/equitorial labelled 5/6 coordinate

 

Heme proteins that bind ligands, like the oxygen transport protein Hemoglobin (Hb), the oxygen storage protein Myoglobin (Mb) and the oxygen sensor protein FixL have a vacant coordination site in the absence of O2. In this case they are only 5-coordinate (Fig. 4A).

When O2 is present it binds to the heme Fe(2+) forming the 6-coordinate, fully ligated protein (Fig. 4B).

Oxygen binding for these heme proteins is typically a reversible, equilibrium process, which can be represented by equations like the following, which is written specifically for Mb.

Mb + O2 = Mb-O2

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I) Heme Ligation is a General Process

 

Many heme proteins are only 5-coordinate in the absence of substrates or ligands. They function by ligand or substrate binding to form 6-coordinate protein. Such ligand binding may not be either reversible or an equilibrium process. For example, peroxidases are thought to function by binding H2O2 at the heme in the earliest steps of their enzymatic cycle, which ultimately results in decomposition of H2O2 into 2 H2O molecules. So, vacant heme coordination sites may also be catalytic sites.

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J) Magnetic Properties, Coordination Number and Structure

For hemes, in proteins and naked in solution, there is an inextricable link between coordination number, the heme molecular structure and heme magnetic properties. This derives from the simple fact that "transition metal chemistry is the chemistry of the d-orbitals"--and the heme iron ion IS a transition metal.

There are five real d-orbitals: dxy, dxz, dyz, dx2-y2, dz2. Under the influence of the "crystal field" of the ligands arrayed around the iron ion these five orbitals split into non-degenerate groups. Two different groupings are shown in Fig. 5. These are characteristic of heme proteins. When the heme iron ion is 5-coordinate the relative energies and groupings are those shown in Fig. 5A. When the heme iron ion is 6-coordinate the groupings and energies are those shown in Fig. 6A.

 

Fig: 5 d orbital diagram A) 5-coord B)6-coord

 

The valence electrons of the heme iron ion are then distributed among these orbitals. The following shows the number of d-electrons for the three oxidation states of Fe:

Fe(2+) = d 6

Fe(3+) = d 5

Fe(4+) = d 4

How these electrons are assigned to the d-orbitals depends upon comparing two fundamental, opposing energies: (1) the energy it takes to pair any two electrons in a single orbital vs (2) leaving them unpaired, but at the increased energy cost of putting one of them in a higher energy orbital. (1) is called the low-spin state, because there is a minimum number of unpaired electrons. (2) is called the high-spin state because it has the maximum number of unpaired electrons.

Fig. 9 shows the known d-orbital electron populations for the three oxidation state of heme Fe. Notice that high-spin configurations occur when the heme iron ion is 5-coordinate, whereas low-spin configurations occur only when the heme coordination number is 6. This is independent of oxidation state (+2 or +3). The (+4) state has only been observed as an oxy-ferryl form, which is 6-coordinate.

 

Fig. 9

d-orbital diagram of electron spin states hs/ls +2, +3, +4. 5coord/6coord.

 

As shown in Fig. 9, seven out of the eight spin/ligation states displayed by heme proteins have unpaired d-orbital electrons. Those four states are paramagnetic. Only the 6-coordinate Fe(2+) state has no unpaired electrons, meaning that it is diamagnetic.

Paramagnetism from unpaired electrons is a physical property of substantial magnitude that can be measured using magnetic susceptibility measurements and allows EPR spectroscopy to be carried out. It complicates NMR spectroscopy. The greater the number of unpaired electrons, the larger is the paramagnetic moment. From Fig. 6, the 5-coordinate, high-spin Fe(3+) state of heme has the largest paramagnetic moment. This, then, is the link between coordination number and magnetic properties stated in the first sentence of this section.

There is a more subtle link between these properties and the actual heme structure. The combination of iron-ion and porphyrin is genuinely planar (or approximately so, including some ruffling of the porphyrin core) only when the heme iron ion is 6-coordinate. When the heme is 5-coordinate the iron ion moves out of the central hole in the porphyrin towards the 5th ligand. Thus, in high-spin, 5-coordinate hemes the iron-ion sits above the heme plane and the porphyrin is domed toward it. These structural changes are shown schematically in Fig. .

 

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