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Chem 32002 ± Fall 2020: Biochemistry article review

1. General information

Student name & email:

Date submitted:

Article reference (including Author names / Title of article / Source (journal / newspaper / site) / Year /

Volume / Page numbers):

Mitra, A., Ko, Y., Cingolani, G. et al. Heme and hemoglobin utilization by Mycobacterium

tuberculosis. Nat Commun 10, 4260 (2019).

Citation DOI: https://doi.org/10.1038/s41467-019-12109-5

2. Summary of article (1500-2000 words):

Mycobacterium tuberculosis (Mtb) is a type of bacteria that is part of the respiratory system which has

a waxy layer because of the mycolic acid present. Majority of the iron that our body stores is found in

heme, which is hydrophobic. Heme has the highest iron source, and it is estimated to have about

70% of the total iron in our body. Iron helps perform a critical function when it comes to taking care of

bacterial infections. There is evidence that shows the high amount of iron can cause a high risk of

Tuberculosis (TB) pathogenesis. Due to gene variation such as haptoglobin, high levels of iron build

up in the blood cell which could lead to an increased risk of TB.

In the event of severe low levels of iron, Mtb secretes siderophores, which are molecules that have

the ability for high iron-binding affinity. It is secreted by bacteria and is used for iron transport through

the cell membrane. It was first proposed that MmpL3 and MmpL11 were responsible for heme

utilization, but it was later found out that they were used as a pump to export lipids as a maintenance

function for the mycobacterial cell wall. It is still unclear whether MmpL3 and MmpL11 do play a role

in the heme utilization by Mtb. It was later discovered that heme-binding proteins present on the cell

surface such as PPE36 and PPE62 were used for heme utilization by Mtb. The study also wanted to

test where PPE36 and PPE62 were responsible for hemoglobin utilization as well. They were placed

in a medium with heme and hemoglobin. Because there was no growth of Δppe36 mutant with heme

or hemoglobin as their only iron source, this tells us that PPE36 is required for both heme and

hemoglobin utilization. Similar results were found for PPE62. When the siderophores deficient mutant

was infected, the survival rate decreased drastically after 72 hours. Based on their study, it showed

that PPE36 was essential for heme utilization and nothing was mentioned about PPE37. But recently

we see that PPE37 is essential and PPE36 is not. The reason for this could be that gene variation

caused changes in the requirements for the Mtb utilization. It was important to note that the study did

not test whether PPE37 played any role in heme and hemoglobin utilization by Mtb.

The study tested whether Dpp transporter plays a part in heme utilization. First, by doing a

homologous recombination, the dpp operon was removed from the Mtb strain. Homologous

recombination is a process of exchanging genetic information between similar nucleic acids. It was

found out that removal of dpp did not affect the growth of the Mtb in Hdb minimal medium which

consisted of 10 µM ammonium ferric acid. But in the presence of 2.5 µM human hemoglobin and 10

µM hemin, it abolished the growth. Ammonium ferric acid, human hemoglobin, and hemin were used

as a source of iron. If in the case where there was some initial growth, it must have been because

dpp operon might not have been completely taken out. To determine whether dpp transporter played

a role in the uptake of heme, wt Mtb, Δdpp mutant, and the complementary strand were monitored.

Dpp mutants are constructed by amplifying the sequence on both sides of the chain with their

corresponding primer pairs. These were then cloned to remove the dpp genes and in order to prevent

polar effects due to deletion, promoters were added. To ensure dpp gene was removed, a

polymerase chain reaction was done. This helped create a large sample of molecules for a

comprehensive study. One of the concerns was that the deletion of dpp operon could change the lipid

composition. This could lead to the barrier being more permeable, which could reduce the heme

uptake. To test the cell membrane permeability of the Δdpp mutant, they introduced the mutant into a

medium containing Ethidium bromide. It was found out that the rates of intake of Ethidium bromide

were both identical for Mtb and the Δdpp mutant. This concluded that the cell permeability did not

have anything to do with the uptake of heme in the Δdpp mutant.

The study wanted to monitor whether peptide played any role in the heme uptake by Mtb. A previous

study showed that peptides inhibited the Dpp-dependent heme utilization in E. coli. In the clinical Mtb

isolate, when the dpp genes were deleted, it caused a toxic reaction with a protein. This tells us that

peptides are involved in dpp transport. After determining that proteins and peptides were involved in

the dpp transport, the team wanted to identify the site where the heme-binding occurred. First, a dppA

with a heme complex was formed. This was then purified through size exclusion chromatography.

Size exclusion chromatography is a way to separate molecules in a solution based on their molecular

weight and size. The proteins were purified by first starting a culture of strains and inserting them into

a medium containing ampicillin. Once the gene expression was induced, they were separated through

a centrifuge. From here, the way to purifying the protein was by adding it to an organic molecule and

washed with a buffer and an eluent. The steps were then repeated several times with a specific

compound until we reach a tiny portion at the end, which was the pure protein. DppA heme was easy

to identify based on their red color. The red color was due to the fractions present in the DppA. In the

size column chromatography, a strong peak was observed at 405 nm.

Figure 1: This figure shows the soret peak of DppA wt at 405 nm, which indicates the characteristic

for heme bound protein. This helps us identify a potential heme-binding site. The x-axis represents

the wavelength, and the y-axis represents the change in absorbance. (Mitra et al., 2019)

At least three crystals were obtained, and they all shared a similar content made up of 34% solvent

and none of them were bound by heme. It was hypothesized that due to the dehydrating agents used

for crystallization, it formed a closed conformation, and this prevented the heme-binding of DppA. If

an excess dehydrating agent were used, it could absorb some of the target compounds. To test the

hypothesis, the surface of the DppA was scanned to determine whether there were any solvent-

accessible pockets. This was done to find out whether there was any heme present or if any histidine

was exposed. Our body can change histidine in various compounds and because histidine plays a

major role in metabolic reactions and supply oxygen to our body, it is the major iron-binding residue in

protein. It was found out that there were indeed pockets present, however, the pockets seem too

small for heme to be present. To examine the pockets, the DppA was put through a normal mode

analysis. It showed that there were large pockets present compared to the scan done previously.

One of the methods used was Ethidium bromide accumulation assay. The reason ethidium bromide

was used because it has an intercalating agent that forms light when it binds to the DNA. This helps

us measure to what degree the accumulation has taken place. First, the strains were grown in a

medium. They were then filtered and allowed the growth to continue to release unnecessary strains.

The cells were then centrifuged, and ethidium bromide was added. Then the fluorescence was

recorded between different intervals. Arginine helps in protein production and because of its amine

side chain, it is found on active sites and therefore arginine 179 helps in the heme-binding process.

It was observed that due to the addition of Bovine serum albumin (BSA), it seemed to affect the

utilization of Heme and Hemoglobin by Mtb. BSA is derived from cows and it consists of 583 amino

acids and it is used in labs as a standard for protein concentration due to its lack of ability to interfere

with reactions. To understand the effect, in a medium of heme and hemoglobin as an iron source,

ppe36 and dpp mutants did not seem to grow. But when BSA was added into the medium, it helps

stimulate the growth of ppe36 and dpp. When the sample was put through an inductively coupled

plasma mass spectrometry, it would that BSA was able to increase the iron concentration by 5 times.

To test whether the iron present in BSA stimulated the growth, they tested the mbtD mutant in two

mediums. One containing no albumin and the other containing 0.5% albumin. Significant growth was

observed in medium with 0.5% albumin, which concluded that albumin can helps the uptake of iron by

Mtb. In one of the experiments, most of the hemin was bound to BSA which has a higher affinity than

the low-affinity PPE36. It was important to note that the mechanism that describes how albumin does

help accomplish significant growth is still unknown.

In conclusion, the study focused on how substrate binding protein DppA, which is part of the Dpp

transport, played a part in the utilization of hemoglobin and heme by Mtb. It also mentions how

albumin skips over the requirements for PPE36, PPE62 and Dpp to bind to heme. The study revealed

that the amount of iron present played an important role in the utilization by Mtb.

3. How does the content/topic of this article relate to biochemistry (200-300 words)?

This article relates to biochemistry in terms of how myoglobin and hemoglobin function, how histidine

plays a role in binding to Fe, and the importance of high and low affinity for heme. In chapter 5, we

went over heme-containing proteins and how myoglobin is responsible for storage and hemoglobin is

responsible for transport. We also learned the function of a ligand and how it can regulate the activity

of the protein by binding which can either regulate or deregulate the function. This can be seen in

how peptides played a role in the uptake of heme by Mtb. We even talked about how proximal

histidine normally binds to Fe, whereas distal histidine does not, and as mentioned above, exposing

histidine can play a critical role in determining where or not heme-binding will occur. We also talked

about how certain membrane-binding proteins can have either a high or low affinity for heme and this

study also mentions that for proper heme utilization to take place, certain substrate-binding proteins

must be present that have a high affinity. The study also mentioned that at a hemin concentration of

10 μM, most hemin was bound to high-affinity BSA and not low-affinity PPE36 and that in the event of

an infection, Mtb uses the albumin pathway for heme uptake.

4. Citations

Mitra, A., Ko, Y., Cingolani, G. et al. Heme and hemoglobin utilization by Mycobacterium

tuberculosis. Nat Commun 10, 4260 (2019).