Give an explanation for the following observations (part d assumes Box 29.2 has been studied):

(a) Both haemoglobin and cytochromes contain haem-iron;

(b) Cytochrome c oxidase contains more than one metal centre;

(c) Each sub-unit in deoxyhaemoglobin contains 5-coordinate Fe(II), but in cytochrome c, the Fe centre is always 6-coordinate;

(d) Nitrophorin (NP1) reversibly binds NO.

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Nitrophorins are haem proteins which are present in the salivary glands of the blood-sucking insect Rhodnius prolixus which lives in South and Central America. Binding of NO to the Fe(III) centre in nitrophorin (NPI) is reversible, and depends on pH. Crucial to the process of blood-sucking by Rhodnius prolixus is the fact that NO binds 10 times more tightly at pH 5 (i.e. the pH of the saliva within the insect) than at pH 7 (i.e. the physiological pH of the victim). Once insect saliva is released into the victim, NO is released, causing expansion of the blood vessels (vasodilation) and inhi- biting blood clotting. In response to being bitten, the victim releases histamine to aid healing of the wound. -0₂C BIOLOGY AND MEDICINE Box 29.2 The specialists: how the blood-sucking Rhodnius prolixus utilizes NO Protein Fe N NH CO₂ NO bound to NP1 (NP1-NO) NH NH₂ Histamine complexed with NO at pH 5.6. The haem unit is bound within a pocket in the protein comprising B-sheets and is tethered to the protein chain by a histidine residue. X-Ray crystallographic data are available on NP1-NO and its [CNJ analogue and confirm that NO and [CNJ bind to the haem Fe. However, in the X-ray beam, photoreduction of NPI-NO readily occurs, and it is difficult to assess whether or not the observed bent coordination mode of the NO ligand is a consequence of photo- reduction. The Fe-N-O bond angles differ between different structural determinations. For the cyanido complex, the Fe-C-N bond angle is 173°. The NO or [CN] ligand is 'lodged' in a pocket of the protein chain between two leucine residues (see Table 29.2). Structural data for the histamine complex show that this same protein pocket hosts the histamine ligand, indicating that NO and histamine compete for the same binding site. At physiological pH, the haem unit in NP1 binds histamine 100 times more strongly than NO. This should both aid the dissociation of NO and inhibit the role of histamine, both of which work in favour of the attacking Rhodnius prolixus.