Early Stages in the Metabolism of Iron 203 



In the titration experiments described above, the two acid groups titrated 

 at low pH can be attributed to protons derived from the carboxyl and the 

 orthophenolic groups. The third proton may arise from either the peptide 

 hydrogen or the meta-hydroxyl. Experiments with space-fiUing models 

 proved that considerable distortion would be required in order to co-ordinate 

 the carboxyl oxygen with the ortho-hydroxyl group of the phenolic ring. 



Paper-chromatographic analyses have revealed that, in addition to itoic 

 acid, several other phenolic acids are produced by B. subtilis in minute 

 quantities. These substances could all be converted to 2:3-dihydroxybenzoic 

 acid by hydrolysis in 6 N HCl and it seems likely that they are conjugates of 

 the single parent phenol with various amino acids and peptides. 



Although neither free itoic acid nor the ferric chelate are particularly 

 stable to decomposition, it can be concluded that their destruction would be 

 negligible under the usual growth conditions required by B. subtilis. 



Of the three possible metabolic mechanisms proposed to account for the 

 appearance of itoic acid in the low-iron growth media, that of the substance 

 acting as a by-pass around the cytochrome system seems to be the most 

 unlikely. This follows from the fact that the relative complexing constant 

 for ferrous ion is no doubt much lower than for ferric ion and also from the 

 fact that there is no known mechanism whereby a cell can obtain energy by 

 transmission of electrons through a quinone-hydroquinone system. The 

 experimental evidence available does not allow a clear-cut choice between the 

 two remaining hypotheses. However, various fragments of information can 

 be pieced together to indicate that the production of itoic acid is the expres- 

 sion of a metabolic block created by the deficiency of iron. Thus iron is 

 known to be required for the activation of ring-opening enzymes, such as 

 catechol oxidase, and it is quite possible that the further metabolism of itoic 

 acid is dependent on the presence of this metal ion. Pre-formed itoic acid is 

 rapidly removed from B. subtilis fermentations following the addition of iron. 

 Further evidence that itoic acid is an emergency agent thrown out to scavenge 

 iron, when this essential element is deficient, comes from the observation that 

 inorganic iron provides a slightly superior rate of initial growth. That a 

 drastic alteration in the carbohydrate metabolism of the organism has 

 occurred is apparent from the observation that the production of itoic acid is 

 accompanied by the formation of large amounts of succinic acid. Thus a 

 portion of the normal intermediates in the carbohydrate metabolism pathway 

 may be forced to terminate at 2:3-dihydroxybenzoic acid which is then 

 excreted as the glycine conjugate. The further metabolism of itoic acid under 

 conditions of normal iron supply may, in fact, be a fairly general reaction in 

 living tissues, including those of the animal organism. For example, admini- 

 stration of as much as 1 g of 2: 3-dihydroxybenzoic acid to a single rat did not 

 lead to the excretion in the urine of a free or conjugated phenol. 



Since itoic acid does not appear to be formed in media containing over 



H.E. — VOL. I — p 



