I was experiencing the same issue: 404 page on comment posting, but additionally, all the comments previously on my site had disappeared, neither listed in dashboard or viewable on the pages themselves.
I tried the methods mentioned: disabling plugins, changing themes, checking permalinks, htaccess rewrite rules, and file permissions, and upgrading WP… none of them worked. It turned out that the problem was my WP comments table had crashed.
It looks like WP-DB Manager Plugin doesn’t have a version for WP 3, so here’s a link on how to fix crashed tables quickly if you have shell access to the wp database files: How to repair a crashed wordpress database
