Bacteriohemerythrin (PDB ID: 4XPX) from Methylococcus capsulatus (Bath)
Created by: Sundus Razzaq
Traditionally found in marine invertebrates, hemerythrins are oxygen carriers that exist in various oligomeric forms with each a monomer containing a highly conserved four-helix bundle structure and a non-heme di-iron center where oxygen binds. A monomeric form of hemerythrin, bacteriohemerythrin from Methylococcus capsulatus (Bath) (PDB ID: 4XPX) was recently discovered and was found to co-express when conditions caused particulate methane monooxygenase (pMMO) in the methanotroph to be overproduced (1). Bacteriohemerythrin, or McHr, was proposed to serve as a transporter for dioxygen from the cytoplasm of the cell to the introplasmic membrane to deliver the dioxgyen to the pMMO enzyme for methane oxidation. Of particular interest was the identification of a water tunnel gated by Leu-114 that is presumed to facilitate autoxidation and the structure of which plays a key role in determining the function of the bacteriohemerythrin (2).
The function of bacteriohemerythrin
can largely be analyzed by discussing the structure that enables the water
tunnel in the methanotroph to exist. Bacteriohemerythrin has a mass of 14,583
Da and an isoelectric point of 5.75, and its crystal structure shows that the structure consists of a single four-a helix bundle with a non-heme di-iron center coordinated by a number of amino acid residues (3). The water tunnel itself is found within the
four-helix bundle, oriented parallel with the long axis of the subunit. The
side chains of the hydrophobic residues His-22, Ile-25, Val-29, Leu-32, Leu-48,
Leu-51, Ile5-2, Val-55, His-58, Phe-59, Glu-62, His-81, Leu-84, Val-87, Leu-91,
Phe-109, Val-110, Trp-113, Leu-114, His-117, Ile-118, and Asp-122, all line the
water tunnel inside the four-helix bundle and the hydrophilic amino acids Cys-88, Gln-92, Thr-106, and Thr-107 are found adjacent to the protein surface between two of the helices and terminate
the water tunnel (2). The fact that the secondary structure consists of a four-helix bundle is very significant because the structure conveniently forms
the shape which allows the water tunnel to exist within it and allow passage of
molecules through. The hydrophobicity of the residues that line the tunnel
serves as a repulsive forces are able to push a water molecule through the
tunnel. A residue of significant functional importance serving this very role in
the water tunnel is Leu-114.
In
various studies and experimentation involving hemerythrins, sequence alignments
of bacteriohemerythrins, a hemerythrin-like domain in Desulfovibrio vulgaris (DcrH-Hr) (PBD ID: 2AVK), and myohemerythrin (PDB ID: 1MHR) show that Leu-114 is
part of the conserved WLVNHI alpha-helical motif that forms the putative water
tunnel (2). Leu-114 serves to control access of a water molecule to the di-iron
core. In another study on water tunnels in DcrH-Hr protein, the side chain of Leu-115 was found to rotate at the end of the tunnel which would open the
channel and enable a water molecule to quickly access the non-heme di-iron
center.Leu-114 is found to behave in much the same way and essentially serves
as a water valve, a hydrophobic barrier that facilities transport of a water
molecule through the tunnel to the di-iron center. Experimentation which
involved comparing the rates of autoxidation of various hemerythrins provides
evidence for Leu-114’s functional role. The autoxidation rate (t1/2)
of bacteriohemerythrin was determined to be approximately 50 minutes, which was
quite longer than the autoxidation of DcrH-Hr, but much faster than the
autoxidation of hemerythrin of marine invertebrates. In comparing specific
mutants Leu-114Y and Leu-114F, in which the side chains were altered to that of
tyrosine’s and phenylalanine’s respectively, to that of the wild type
bacteriohemerythrin, the autoxidation of the mutants was found to be slower and
much more similar to that of the hemerythrin from the marine invertebrates (2).
The longer autoxidation of the mutants suggests the role that the particular
leucine plays as the hydrophobic barrier.
Another
significant feature of the structure of McHr is its active site, the non-heme di-iron core found at the end of the water tunnel. As the name suggests, the
di-iron site contains two Fe (II) ligands which serve to bind oxygen molecules
for the process of autoxidation. One of the iron atoms, Fe1, is six-coordinated with His-77, His-81, His-117, Asp-122 and Glu-62. The second iron atom, Fe2, is associated
with a hydroxide and the protein ligands His-22, His-58, Asp-122 and Glu-62 (2).
While properties of the water tunnel and even its presence or lack thereof may
vary between bacterial hemerythrins and invertebrate hemerythrins due to
various required functions such as storage or dioxygen delivery, the structure
of the di-iron core is generally conserved in various species. In its
deoxygenated state, bacteriohemerythrin, as well as other hemerythrins, the two
iron atoms are bridged by a hydroxide and after oxygenation, the proton is
transferred to the bound dioxygen and a hydroperoxo ligand is formed (4). After
the core has been exposed to water for an extended period of time the di-iron
site undergoes auto-oxidation and the oxidized state of the
bacteriohemerythrin, in which there is no bound dioxygen, is produced.
In
analyzing the how the structure of bacteriohemerythrin allows for it to
properly function in autoxidation, comparisons to a similar protein found
through PSI-BLAST and Dali searches can provide more insight into the functions
that distinguish the protein of interest from others. The purpose of the PSI-BLAST
is to search for similar structures, subjects, to the protein query, which in
this case was bacteriohemerythrin. Proteins of similar structure are assigned
an E-value which is calculated by the total sequence homology and by assigning
gaps which are amino acids found in the subject’s sequence but not in the
query’s sequence. An overall high sequence homology corresponds to a low
E-value, which is considered to be significant if below 0.05, while gaps increase
the E-value. The Dali Server was used to find proteins with tertiary structures
that resemble the specific protein being analyzed, bacteriohemerythrin. The
server implements a sums-of-pairs method, comparing intramolecular distances
that produce a measure of similarity represented by a Z-score. A Z-score above
2 indicates that the structures have significant similarities. The PSI-BLAST
showed a protein, methemerythrin (PDB: 1I4Y), similar to bacteriohemerythrin but derived from the organism Phascolopsis gouldii, with an E-value of 6e-36 (5). From the Dali Server search, the
Z-score for this protein was found to be 16.3 (6). Other than the proteins
being found in different organisms, there are also some interesting structural
differences between the two proteins. The methemerythrin is a homooctamer with each of its 8 subunits consisting of a four-helical bundle. There is no water
tunnel present in this protein, as are absent from hemerythrins form marine
invertebrates (6). There is also the presence of another ligand, Cl-,
which has no interactions at the active site of the iron atoms, but may be
involved with the dissociation behavior of its oxygenated form (7). The comparison
protein’s function is also to serve as a dioxygen transporter to the di-iron
core, which is ubiquitous in hemerythrins, but there are slight differences in
some of the molecules involved for autoxidation. The protein consists of a
Fe-O-Fe ligand in its core, but the crystal structures show that the hydrophobic
binding site is able to tolerate the substitution of a normally present leucyl
side chain with a tyrosyl side chain without causing any significant structural
reconfigurations anywhere else. In addition, the binding pocket favors
localization of the tyrosyl hydroxyl side chain near the dioxygen binding site.
This interaction allows for a stabilizing interaction between the tyrosyl
hydroxyl and the oxygenated form of the methemerythrin as close proximity
allows the hydroxyl group to donate a H atom to the hydroperoxo ligand (6).
Analysis
of the structure of bacteriohemerythrin provides insight for its function as an
oxygen transporter, with the 4 helical bundle itself serving a scaffoldind, a
structural fold in which a water tunnel can exist. The packing of the alpha
helical motif with the hydrophobic barrier of Leu114 controls access of water
to the di-iron core. Comparing the structure of bacteriohemerythrin to other
proteins elicits the comparison of the functions that are dependent on these
specific structures. Hemerythrins from marine invertebrates are able to carry
out autoxidation without the need of a water tunnel which is present in
bacteriohemerythrins. Research has shown that for a case when dioxygen is not
consumed by pMMO for a long period of time, if there was, for example, no
hydrocarbon available to the pMMO, the oxygenated form of bacteriohemerythrin
would be converted to another form in which it could be released to the cytosol
for re-oxidation in another cycle of dioxygen transport (2). The various
functions found in hemerythrins illustrates how the slight alterations of
mostly similar structures underlying all these proteins allows for a diverse
array of proteins to function under unique conditions as exemplified by
bacteriohemerythrin.