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As it says in the "Searching the Index ... Limitations" section about the Colonial Secretary's Letters "The index is not a complete list of all material within the series NRS 905". Perhaps an email to the NSW Archives might help? Looks like you need Reel 2934 Item (5/2400) .
https://mhnsw.au/articles/922/

The reel you mention is just an Index to the register of letters received, ie it gives a surname or subject and then the reference number, nothing more.  You then need to go to the actual register of letters received to get more details, which I've already done - 44/2540 was dated 28 Mar 1844 from the Royal Engineers, ie Appl[ication]n from Lt Hamilton RE Norfolk Island for use of Govern[men]t House

I've been through the Index for the letter M, Jan-Jun 1844, and there's nothing at all for Mackley or anything similar.

I also looked up 44/2546 in case it was bad handwriting, but this relates to John McCurdy of Penrith regarding his wife Sophia, and is dated 26 Mar 1844.

I think you could be right though and that an email to the archives may be needed.

Thanks also to Christine for the Cecil Hills info - I'd done a little bit of searching but had planned to come back to it, so hopefully the database will be up and running again by then 

There was another convict John Mackley, came out on the David Clarke in 1841, so that 1844 letter may be his:

I did consider that and then dismissed it as the annotation seemed very specific to the Earl Spencer, but having looked again there's something in the Hobart Town Gazette, 1844 (I'll have to dig around for a more specific date) saying:

John Mackley, David Clarke, to ditto (to Allan M'Kinnon, Dalness)

But then what has 1838 got to do with anything if this man only arrived in 1841?

The reference 44/2540 in the register of letters received was dated 28 Mar 1844 from the Royal Engineers and says (I think!): Appl[ication]n from Lt Hamilton RE Norfolk Island for use of Govern[men]t House, so doesn't seem to connect to either man!

No 44/2540 dated 6 Mar 1844 has to refer to something, but what?  And where?

Another dead end then by the look of it.

I've been trying to figure out that reference to the letter - 44/2540 dated 6 Mar 1844 and it seems to be referenced here under Reel 2537 then either item 2401 or 2403

https://mhnsw.au/articles/922/#Indexes-1826-1906

I've looked on Ancestry on the Register of Letters Received 1843-1845 and there is an entry numbered 44/2540 (image 233), but its nothing to do with John Mackley and not dated 6 March 1844, so I'm more confused than ever 

Ooh now that's interesting.  He did have a son named John who became an ag lab, but I'm not aware of one named William so I'll look into that. 

Ann, his wife, died just a few months after he was imprisoned so I've also been looking for any records relating to the children in this period, but so far I haven't found anything.

EDIT: Just seen your edit.  Oh well!