s/(two )(?!.*two )//
[download]

Probably valid only perl 5.6.0 +. Update: Above works only if two is followed by a space. It fails if:

$string = "one two three two four two";
[download]

The following works. Also, the parentheses around the first two are unnecessary.

$string =~ s/two\s*(?!.*two)//
[download]