Shepherd Lecture 2 - Nitric Oxide

Front 1

Outline how nitric oxide causes damage to bacteria

Back 1

NO can diffuse across the bacterial membrane and react with haem groups, thiols and destroy iron sulphur clusters

NO can damage respiratory cytochromes which causes respiratory inhibition

NO can react with superoxide produced by NADPH oxidase to form peroxynitrite which can nitrate protein residues

Front 2

When does bacteria encounter NO?

Back 2

Dietary nitrites react with stomach acids which causes the formation of nitric oxide

Gut bacteria respire anaerobically, nitric oxide is an intermediate of anaerobic respiration. Nitrate/nitrite is used as an electron acceptor which can get reduced to nitric oxide

Commensal bacteria produce NO

iNOS produce NO during infections

eNOS

Front 3

How do most nitric oxide synthases work?

Back 3

Arginine --> Hydroxyarginine --> Citrulline + nitric oxide

Front 4

Describe the structure of LPS

Back 4

A core which is connected to an O-specific chain and Lipid A (two glucosamine residues bound to six fatty acids)

Front 5

How does the body detect gram negative bacteria?

Back 5

Using its O-specific chain

Front 6

Draw a diagram of inducible nitric oxide synthase (iNOS)

Back 6

Front 7

Draw a diagram demonstrating how NO is produced by iNOS

Back 7

Front 8

True or false? CaM is activated by Ca2+

Back 8

No, it's calcium insensitive but other isoforms of NOS is activated by Ca2+

Front 9

What makes CaM calcium insensitive?

Back 9

CaM is covalently bond between calmodulin and calcium

Front 10

In other NOS isoforms, what does NO do?

Back 10

Nitric oxide stimulates guanylate cyclase which increases cGMP levels. This stops calcium entry into the cell which results in diminished eNOS dependent nitric oxide production

Front 11

What adaptations does E.coli have which helps it to resist nitric oxide?

Back 11

1. Flavohaemoglobin hmp - converts nitric oxide to nitrate (aerobic) and into nitrous oxide (anaerobic)

2. Flavorubredoxin reductase NorVW - nitric oxide converted to nitrous oxide during anaerobic conditions

3. Cytochrome nitrite reductase NrfA - NO reduced

4. Diiron protein YtfE - repairs damaged cytochromes caused by NO damage

5. Cytochrome bd1 - Is a NO-tolerant respiratory oxidase which helps to carry on aerobic respiration

Front 12

What is cytochrome bd-1?

Back 12

It's a NO-tolerant final electron acceptor which reduces oxygen into water. Transfers 2H+ from QH2 into the periplasm which creates a proton gradient which ATP synthase uses to generate ATP

Front 13

What does flavohaemoglobin consist of?

Back 13

FAD-binding domain, NAD-binding domain and a globin

Front 14

How does cytochrome bd-1 help against NO?

Back 14

Normally, NO damages respiratory cytochromes which means it can't generate ATP.

Cytochrome bc-1 is NO-tolerant meaning that NO doesn't affect it, meaning that it can still produce ATP

Front 15

Outline the reaction for flavohaemoglobin under aerobic and anaerobic conditions

Back 15

Front 16

What catalyses the flavohaemoglobin hmp reaction?

Back 16

The haem cofactor on the flavohaemoglobin hmp

Front 17

What happens to the haem cofactor during the reaction?

Back 17

The reaction converts the haem cofactor from the ferrous (Fe2+) to the ferric (Fe3+) form. This is done by oxidising an NAD in the NAD binding site. An electron is donated and moved to the globin area through the FAD co-factor.

Front 18

Outline the two ways hmp converts NO to nitrate

Back 18

Front 19

Explain the two ways hmp converts NO to nitrate

Back 19

In the early phases of an infection, when lots of oxygen is present and very little NO, they dioxygenase reaction is used. This is when oxygen binds first to convert Hmp-Fe(II) to Hmp-Fe(II)O2, using NO and converting it to NO3-. This produces Hmp-Fe(III) which is then reduced back to Hmp-Fe(II)

In the late phases of infection, when lots of NO is present produced by immune cells, the dinitrosylase reaction is taken. This is in which NO binds to Hmp-Fe(II) which turns into HMP-Fe(II)NO. Oxygen is then used to convert the NO to NO3- which produces Hmp-Fe(III), which in turn is reduced back to Hmp-Fe(II).

Front 20

Name two ways hmp is regulated under aerobic conditions

Back 20

NsrR AND MetR

Front 21

How is hmp regulated under anaerobic conditions?

Back 21

FNR

Front 22

Explain how hmp is regulated under aerobic conditions?

Back 22

NsrR -> normally represses hmp transcription, in the absence of NO. When NO is present, the iron-sulphur clusters in NsrR is nitrosylated which causes a loss of DNA binding, damaging NsrR so it can no longer regulate the transcription of hmp, causing hmp to be expressed

MetR -> MetR activates hmp transcription. MetR is regulated by homocysteine. When NO is present, homocysteine is damaged and reduced meaning MetR is regulated less. This leads to the transcription of more Hmp genes.

Front 23

Explain how hmp is regulated under anaerobic conditions

Back 23

FNR -> NO reacts with the iron-sulphur clusters of FNR. The iron-sulphur centres usualy repress the hmp gene. When NO is present, it damages iron-sulphur clusters which deactivates FNR. This leads to hmp no longer being repressed.

Front 24

Which type of bacteria produce nitric oxide?

Back 24

Gram-positive bacteria producing NO are called bNOS

Front 25

What do bNOS lack?

Back 25

A reductase domain

Front 26

How is NO produced to give bacteria an advantage?

Back 26

It gives bacteria resistance to a variety of antibiotics

Front 27

How are NO-induced advantages achieved?

Back 27

1. Chemically altering NO to less toxic compounds

2. Getting rid of oxidative stress imposed by many antibiotics

3. NO suppresses the antibiotic-mediated formation of reactive oxygen species.